Blood Advances

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6. To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.

We employ and extensively characterise an ex vivo culture system to study terminal erythroid maturation of CD34+ progenitors from the peripheral blood of normal individuals and patients with Congenital Dyserythropoietic Anaemia type 1 (CDA-I). Using morphological analysis, FACS analysis and the proteomic approach CyTOF, we analysed patient-derived erythroblasts stage-matched with those from healthy donors during the expansion phase and into early differentiation. In patient cells, aspects of disordered erythropoiesis manifest midway through differentiation, including increased proliferation and changes in the DNA accessibility profile. We also show that cultured erythroblasts from CDA-I patients recapitulate the pathognomic feature of this erythroid disorder with up to 40% of the cells having abnormal spongy chromatin morphology by electron microscopy, as well as upregulation of GDF15, a marker of ineffective erythropoiesis. In the tertiary phase of culture, patient cells show significantly less enucleation and there is persistence of earlier erythroid precursors. Furthermore, the enucleation defect appears to be more severe in patients with mutations in C15orf41, as compared to the other known causative gene CDAN1, indicating a genotype/phenotype correlation in CDA-I. Such erythroblasts are a valuable resource for investigating the pathogenesis of this disease and provide the opportunity for streamlining diagnosis for CDA-I patients and ultimately other forms of unexplained anaemia.

To explore the role of clonal hematopoiesis (CH) on chimeric antigen receptor (CAR) T-cell therapy outcomes, we performed targeted deep-sequencing on 114 large B-cell lymphoma patients treated with anti-CD19 CAR T-cells. We detected CH in 42 (36.8%) pre-treatment patient samples, most frequently in PPM1D (19/114) and TP53 (13/114) genes. The incidence of grade [≥]3 immune-effector cell-associated neurotoxicity syndrome (ICANS) was higher in CH-positive patients compared to CH-negative patients (45.2% vs. 25.0%, p=0.038). Higher toxicities with CH were primarily driven by three CH genes, DNMT3A, TET2 and ASXL1 (DTA mutations). The incidence of grade [≥]3 ICANS [58.9% vs. 25%, p=0.02] and grade [≥]3 cytokine release syndrome [17.7% vs. 4.2%, p=0.08] were higher in patients with DTA mutations than those without CH. The estimated 24-month cumulative incidence of therapy-related myeloid neoplasms after CAR-T therapy was higher in patients with CH than those without CH (19% [95%CI: 5.5-38.7] vs. 4.2% [95%CI: 0.3-18.4], p=0.028). Statement of SignificanceOur study reveals that clonal hematopoiesis mutations, especially those associated with inflammation (DNMT3A, TET2, ASXL1), are associated with severe grade toxicities in lymphoma patients receiving anti-CD19 chimeric antigen receptor therapy. Further studies to investigate the mechanisms and interventions to improve toxicities in the context of CH are warranted.

BackgroundSezary syndrome (SS) is a rare and aggressive leukemic variant of cutaneous T-cell lymphoma (CTCL) with limited therapeutic options and a median survival of fewer than five years. Despite advances in how single-cell RNA sequencing (scRNA-seq) has improved the understanding and treatment of other cancer types, such insights in SS remain limited. The drivers of disease progression and immunologic dysfunction are incompletely defined, underscoring the need to characterize both malignant T cells (MTCs) and their interactions with surrounding immune populations. ObjectivesTo systematically characterize malignant and non-malignant immune cells in CTCL, identify distinct malignant T-cell subtypes, and uncover transcriptional programs and immune evasion pathways with therapeutic relevance. MethodsWe analyzed scRNA-seq data on peripheral blood mononuclear cells (PBMCs) from 22 SS patients and 7 healthy controls. MTC subtypes were identified using a combination of transcriptional profiling, copy number variation (CNV) analysis, and T-cell receptor (TCR) clonotyping. CITE-seq was utilized on a subset of samples to correlate genetic findings with surface protein expression. ResultsWe identified three MTC subtypes: (1) MTC central memory (CM), a Th2-skewed CM phenotype that constituted the dominant malignant population; (2) MTC effector/effector-memory (E/EM), a subset enriched in Th1-associated genes; and (3) MTC regulatory (Reg), a regulatory-like, exhausted phenotype, along with shared and subtype-specific gene signatures. The predominance of MTC CM suggests a stable malignant state, while relative rarity of MTC E/EM and MTC Reg may reflect treatment effects or disease progression. In addition to KIR3DL2, we identified KIR2DL3 and KIR3DL1 as upregulated immune-evasive receptors on MTCs and surrounding cells. Tensor factorization of ligand-receptor interactions revealed pro-inflammatory yet immunosuppressive signaling in myeloid cells converging on STAT3 activation. ConclusionsThis study defines three transcriptionally and functionally distinct MTC subtypes in SS, highlighting subtype-specific vulnerabilities that may inform personalized treatment strategies. Our findings suggest that targeting not only MTCs but also KIR-family receptor signaling and JAK-STAT activation in the immune microenvironment may have therapeutic implications. The identification of novel immunosuppressive pathways and cell survival mechanisms opens avenues for tailored interventions - including widespread KIR inhibition, repurposing JAK inhibitors, and other novel therapies - to improve patient outcomes in SS. Contributor StatementB.A. Childs conducted the conceptualization (equal), data curation (equal), formal analysis (lead), investigation (lead), methodology (equal), validation (equal), visualization (equal), writing - original draft (lead), writing - review & editing (supporting). E. Elghonaimy provided conceptualization (equal), data curation (equal), formal analysis (equal), investigation (equal), methodology (equal), resources (equal), supervision (equal), validation (equal), visualization (equal), writing - original draft (supporting), and writing - review & editing (supporting). P. R. Geethakumari provided data curation (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). K. Kumar contributed conceptualization (supporting), data curation (supporting), resources (supporting), and writing - review & editing (supporting). J. F. Merola provided conceptualization (supporting), investigation (supporting), resources (supporting), writing - review & editing (supporting). H.W. Goff contributed conceptualization (equal), data curation (equal), formal analysis (supporting), funding acquisition (lead), investigation (supporting), methodology (supporting), resources (supporting), supervision (equal), visualization (equal), writing - review & editing (equal). T. A. Aguilera provided conceptualization of the project (equal), data curation (equal), formal analysis (equal), funding acquisition (equal), investigation (equal), methodology (equal), project administration (equal), resources (lead), supervision (lead), visualization (supporting), writing - original draft (supporting), writing - review & editing (equal). T.A. Aguilera is the guarantor and accepts full responsibility for the overall content, had access to all the data, and controlled the decision to publish. Bulleted StatementsO_ST_ABSWhat is already known about this topic?C_ST_ABSO_LISezary syndrome is a rare, aggressive CTCL subtype associated with poor survival; a minority of patients achieve long-term remission with allogeneic stem cell transplant. C_LIO_LIKIR3DL2 is an investigational target with ongoing trials (e.g., lacutamab, anti-KIR3DL2 agent). C_LIO_LIJAK inhibitors have shown mixed effects, with anecdotal benefit and potential harm. C_LIO_LIThe heterogeneous biology of Sezary cells remains poorly characterized, limiting diagnostic and therapeutic advancement. C_LI What does this study add?O_LIWe define three distinct malignant T-cell subtypes in Sezary syndrome, each with clinical and therapeutic implications, and a unifying genetic signature. C_LIO_LIIn addition to KIR3DL2, a known CTCL marker, we identify KIR2DL3 and KIR3DL1, inhibitory receptors implicated in viral and autoimmune regulation, as additional immune evasion mechanisms. C_LIO_LIJAK/STAT activation in neighboring myeloid cells may be driven by Sezary cell signaling through S100A8, S100A9, CD74, IL-10, and TNF, a potentially targetable axis. C_LI What is the translational message?O_LIThe predominance of a central memory-like malignant phenotype, alongside other distinct subsets reveal an opportunity for subtype-guided treatment strategies. C_LIO_LIOptimal therapeutic targeting may include both malignant T cells and interacting immune cells. C_LIO_LIOur findings identify targets that may be actionable, including KIR-family receptors and STAT3-related signaling. C_LIO_LIInsights from other T-cell disorders may guide repurposing of existing therapies to improve outcomes. C_LI Lay Summary: How Sezay Syndrome, a Type of Skin Lymphoma, Impacts the Immune SystemSezary syndrome (SS) is a rare and aggressive form of skin cancer that starts in the blood. It is a type of cutaneous T-cell lymphoma (CTCL), where cancerous T cells (a kind of white blood cell) spread from the bloodstream to the skin. SS mainly affects adults and has limited treatment options, with most people living less than five years after diagnosis. This study, based in the United States, aimed to understand how cancerous T cells behave in SS and how they interact with other immune cells. We used a method called single-cell RNA sequencing to examine over 100,000 individual immune cells from the blood of 22 patients with SS and 7 healthy individuals. This allowed us to see which genes were active in each cell. We found that the cancerous T cells in SS are not all the same. Instead, they fall into three distinct groups: one resembled central memory T cells that typically live in the blood, one looked more aggressive and inflammatory, and one resembled exhausted regulatory immune cells. We also discovered that cancer cells and surrounding immune cells expressed molecules called KIRs, which may help them avoid being attacked. Other immune cells released signals that turned on a pathway called JAK-STAT, which may further protect the cancer. These findings reveal new ways that SS protects itself and avoids the immune system. Understanding these escape routes may help guide future treatments, including drug combinations that block these signals and improve patient outcomes. Abbreviated AbstractSingle-cell RNA and protein profiling of Sezary syndrome reveals three distinct malignant T-cell subtypes and uncovers convergent immunosuppressive signaling through KIR-family receptors and STAT3 activation. These findings expose targetable pathways driving immune evasion and offer a foundation for more personalized therapeutic strategies in this aggressive form of cutaneous T-cell lymphoma.

Syndecan 2 (Sdc2) is a transmembrane cell-surface heparan sulfate proteoglycan (HSPG) that has been implicated in the regulation of cell-cell signaling pathways and cell-matrix interactions. Surprisingly, homozygous recessive maternal zygotic (MZ) sdc2 null mutants in zebrafish appear to have normal development, normal morphology and are viable and fertile in adulthood. Whole transcriptome RNA sequencing, FACS analyses, and imaging of transgenic reporter lines that distinguish specific hematopoietic lineages revealed that sdc2 mutants have defects in the specification and proportions of red blood cells and neutrophils that initiate during embryonic hematopoiesis and likely persist through adulthood. During bacterial infections, MZsdc2 mutants have markedly reduced neutrophil recruitment and significantly higher death rates. Hematopoietic stem/progenitor cell (HSPC) numbers are also significantly reduced in MZsdc2 mutants. In zebrafish, cells that bud off of the ventral region of somites are thought to give rise to the reticular stromal cells of the caudal hematopoietic tissue (CHT) stem cell niche. In MZsdc2 mutants, these budding cells have abberant blebbing morphology associated with widespread apoptosis during induction of HSPCs and with changes in the vascularization and stromal cell structure of the CHT stem cell niche. This suggests that loss of sdc2 disrupts the earliest events of definitive hematopoiesis. Our findings of hematopoietic defects, nascent immune system alterations and inability to resolve infection in sdc2 mutants sets the stage for examining the roles of HSPG genes in a wide range of hematopoietic and immune defects in humans. Key PointsO_LISyndecan 2 regulates the formation of Hematopoietic Stem/Progenitor Cells, differentiation into hematopoietic populations and the CHT architecture C_LIO_LISyndecan 2 mutants are significantly more susceptible to bacterial infection C_LI

Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by excessive proliferation of myeloid lineages. Accurate classification and appropriate management of MPNs requires integration of clinical, morphological and genetic findings. Despite major advances in understanding the molecular and genetic basis, morphological assessment of the bone marrow trephine (BMT) remains paramount in differentiating between MPN subtypes and reactive conditions. However, morphological assessment is heavily constrained by a reliance on subjective, qualitative and poorly reproducible criteria. To address this, we have developed a machine-learning strategy for the automated identification and quantitative analysis of megakaryocyte morphology using clinical BMT samples. Using a sample cohort of recently diagnosed or established ET (n = 48) and reactive control cases (n = 42) we demonstrated a high predictive accuracy (AUC = 0.95) of automated tissue ET diagnosis based upon these specific megakaryocyte phenotypes. These separate morphological phenotypes showed evidence of specific genotype associations, which offers promise that an automated cell phenotyping approach may be of clinical diagnostic utility as an adjunct to standard genetic and molecular tests. This has great potential to assist in the routine assessment of newly diagnosed or suspected MPN patients and those undergoing treatment / clinical follow-up. The extraction of quantitative morphological data from BMT sections will also have value in the assessment of new therapeutic strategies directed towards the bone marrow microenvironment and can provide clinicians and researchers with objective, quantitative data without significant demands upon current routine specimen workflows.

Severe injuries result in acute changes in platelet number and function, but the impact of trauma and hemorrhagic shock on megakaryocytes (MKs) in the initial hours after injury have not been studied in detail. Using a murine model of trauma-hemorrhage, we identified rapid changes in MK morphology and mobilization into bone marrow sinusoids, changes that were detectable within one hour. Levels of several alpha-granule derived proteins were elevated in the bone marrow, and co-culture of naive MKs with bone marrow supernatant from injured mice resulted in similar changes to those observed in the model. These results illustrate that trauma-hemorrhage results in a hyperacute alteration in the bone marrow micro-environment that alters MK activity within an hour of injury.

Platelet -granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germline RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMM), is associated with thrombocytopenia, platelet dysfunction and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen and IgG levels were decreased in a FPDMM patient. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, siRNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared to control cells, with increases in caveolin-1 and flotillin-1 (two independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes) and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 knockdown increased colocalization of albumin with flotillin and of fibrinogen with RAB11 suggesting altered trafficking of both. The increased albumin and fibrinogen uptake and levels of caveolin-1, flotillin-1, LAMP2 and IFITM3 were recapitulated by shRNA RUNX1 knockdown in CD34+-derived MK. These studies provide the first evidence that in RUNX1-haplodeficiency platelet endocytosis of albumin and fibrinogen is impaired and that megakaryocytes have enhanced endocytosis with defective trafficking leading to loss of these proteins by distinct mechanisms. They provide new insights into mechanisms governing endocytosis and -granule deficiencies in RUNX1-haplodeficiency. Key pointsO_LIPlatelet content and endocytosis of -granule proteins, albumin, fibrinogen and IgG, are decreased in germline RUNX1 haplodeficiency. C_LIO_LIIn RUNX1-deficient HEL cells and primary MK endocytosis is enhanced with defective trafficking leading to decreased protein levels. C_LI

Thrombocytopenia is a common and serious complication of anticancer therapies. Here, we identify a novel thrombopoietic activity of the first-in-class ribosome biogenesis inhibitor CX-5461. In a phase I trial, 56% (9/16) of patients exhibited up to a 34% increase in platelet count following a single dose of CX-5461. In mice, CX-5461 elicited a rapid, reversible, and sustained [~]1.7-fold increase in platelet numbers without altering platelet function, lifespan, or inflammatory cytokines. Bone marrow analysis revealed a specific expansion of megakaryocytes (MKs), increased Sca1 MKs, and selective enrichment of MK-biased multipotent progenitor 2, independent of thrombopoietin (TPO) or c-mpl signalling. CX-5461 also mitigated carboplatin-induced thrombocytopenia, accelerating platelet recovery. Single-cell RNA sequencing and RNA velocity analysis confirmed enhanced differentiation of MK progenitors. These findings demonstrate that inhibition of ribosome biogenesis promotes TPO-independent megakaryopoiesis and identifies a previously unrecognised therapeutic opportunity to support platelet recovery in cancer treatment and potentially other thrombocytopenic states.

Understanding how in vivo platelet biogenesis is undertaken is critical to making on-demand platelet production for clinical use feasible. We recently described the discovery of plasma membrane budding as a major in vivo platelet-producing pathway. In vitro recapitulation of this finding could pave the way towards efficient laboratory-based platelet production. The plausibility of the plasma membrane budding model has been called into question. The foundation of this is the contention that the size and payload composition of plasma membrane buds are not consistent with bona fide platelets. Thus, membrane buds likely represent stages in megakaryocyte-derived microparticle formation. Using 3D super-resolution imaging, we have performed a quantitative comparison of size and organelle content of plasma membrane buds, platelets, and microparticles in the adult mouse bone marrow. We unequivocally demonstrate that the structures we previously described as membrane buds exhibit the same size range as free platelets, that all buds contain organelles, and that membrane buds and free platelets contained an equivalent number of organelles. Crucially, membrane buds and microparticles are completely distinct from each other. To prevent future confusion between the processes of microparticle formation and platelet biogenesis, we propose using the more specific term "pre-platelet membrane buds".

Hematopoietic stem and progenitor cell (HSPC) niches support lifelong production of blood and immune cells. Recently, we identified a gene signature unique to HSPC niche endothelial cells that is highly conserved across species and developmental time, and includes the scavenger receptors, stab1/2 and mrc1a. Whether these receptors support HSPC development in the niche remains unclear. To investigate this, we used chemical inhibition and CRISPR mutagenesis in zebrafish and found that loss of stab2, and to a lesser degree stab1, reduced the number of embryonic HSPCs labeled by runx1:mCherry. Subsequent analyses of runx1:mCherry; cd41:GFP double transgenic embryos revealed an imbalance in the HSPC pool in stab2 mutants, with reductions in sub-populations containing stem cells and erythroid progenitors, the latter of which was most decreased. Our findings suggest stabilin scavenger receptors support HSPC development in the fetal niche, which could inform clinical strategies for culturing and expanding HSPCs.

Diffuse large B-cell lymphoma (DLBCL) is characterised by pronounced genetic and biological heterogeneity. Several partially overlapping classification systems exist - developed from mutation, rearrangement or gene expression data. We apply a customised network analysis to nearly five thousand DLBCL cases to identify and quantify modules indicative of tumour biology. We demonstrate that network-level patterns of gene co-expression can enhance the separation of DLBCL cases. This allows the resolution of communities of related cases which correlate with genetic mutation and rearrangement status, supporting and extending existing concepts of disease biology and delivering insight into relationships between differentiation state, genetic subtypes, rearrangement status and response to therapeutic intervention. We demonstrate how the resulting fine-grained resolution of expression states is critical to accurately identify potential responses to treatment. Significance statementWe demonstrate how exploiting data integration and network analysis of gene expression can enhance the segregation of diffuse large B-cell lymphoma, resolving pattens of disease biology and demonstrating how the resolution of heterogeneity can enhance the understanding of treatment response.

Developing novel therapeutics requires robust early-stage target de-risking to ensure safety and efficacy. We developed a scalable proteogenomic framework integrating population-scale human genetics and plasma proteomics to identify genes tolerant of inactivation (i.e., dispensable) within hematopoietic compartments, thereby enabling safer targeted immunotherapies. Using CD33 as a validated benchmark, we observed that naturally occurring loss-of-function (LoF) variants lead to concordant RNA and protein depletion, supporting functional gene inactivation. Early clinical results from the Trem-Cel trial (NCT05945849) further provide proof of concept that deletion of dispensable antigens can enable safe and effective immunotherapy in humans. We extended this approach genome-wide in the UK Biobank and identified 237 candidate dispensable genes, filtered by plasma proteomic data and hematopoietic expression, highlighting LY75 (CD205) as a novel candidate with strong proteogenomic evidence of LoF tolerance. This work establishes a generalizable, quantitative proteogenomic framework for systematic prioritization of dispensable gene targets for editing, providing a foundation for next-generation cell and gene therapies that minimize on-target, off-tumor toxicities.

To-date the genomic landscape of cutaneous pericytic tumours (PTs), which represent a morphological continuum, have not been comprehensively explored. In order to identify the driver events of PTs from across their histological spectrum, and potentially aid the current classification system, we sequenced DNA (whole-exome) and RNA (pulldown transcriptome) from tumour-normal pairs classified by two different dermatopathologists of angioleiomyoma (n=37), glomus tumour (n=30) and myopericytoma (n=11); with all sequencing data deposited in the European Genome and Phenome Archive.

Acute myeloid leukaemia (AML) is a highly heterogeneous disease. Here, we decipher the disease composition of a single AML patient through longitudinal sampling scrutinized by high-resolution genetic and phenotypic approaches, including sequencing, immunophenotyping, ex vivo drug sensitivity testing and establishment of patient-derived xenograft models. Throughout the disease course we identified patterns of both divergent and convergent molecular evolution within the leukemic compartment. We identified at least six discrete leukaemia initiating cell populations, of which five were characterised by known recurrent mutations in AML. These populations partly correlated with immunophenotypically defined cell subsets, drug sensitivity profiles and population-specific potential for engraftment in immunodeficient mice. Our results indicate that the genetic and phenotypic development are closely intertwined, and that diversity in the leukaemic gene-environment likely influences disease trajectories. SIGNIFICANCENovel therapeutic approaches in AML are characterised by targeting molecular mechanisms thought to drive leukemogenesis, but emergent evidence suggests that intra-leukemic composition may be more diverse than previously appreciated. Through in-depth genetic and phenotypic characterization of the disease course of a single AML patient, we demonstrate a high degree of inter-individual complexity that exceeds the prevailing disease conception. The temporal molecular landscape of this patient suggests that leukemogenic transitions may not be categorically monoclonal. Patterns of converging molecular evolution further imply that higher levels of biological organisation than the molecular machinery of single cells may influence leukemogenic trajectories. Disease dynamics, relational properties and causal contribution from several levels of biological organization comes into conflict with the linear monocausal explanatory model on which precision oncology is largely built. This may have implications for current precision oncology oriented prectices, including molecular categorization, molecular therapeutic targeting and predictive models.

Patients with BRCA1/2-mutated ovarian, breast, prostate, or pancreatic tumors can be treated with poly ADP-ribose polymerase (PARP) inhibitors. PARP inhibitors, however, are known to cause therapy-related myeloid neoplasms (t-MN) in a subset of patients. Predisposing factors to t-MN development in the context of PARP inhibitor exposure are not well described. To determine the frequency of t-MN in these patients, an institutional cohort of 265 patients with exposure to PARP inhibitors was identified. A subset of these patients with PARP inhibitor-related cytopenias underwent bone marrow biopsies. Among 265 patients, 17 (6.4%) underwent a bone marrow biopsy, which yielded a therapy-related hematologic diagnosis in 47% (8/17). Breast cancer metastasis to the marrow was found in one patient, and hemophagocytic lymphohistiocytosis was found in another. We analyzed the molecular characteristics of t-MNs in 13 PARP inhibitor-exposed patients, including five additional PARP inhibitor-exposed patients diagnosed with t-MNs in community practices. Among patients with t-MNs, five had acute therapy-related myeloid leukemia (t-AML), six were diagnosed with therapy-related myelodysplastic syndrome (t-MDS), and two had therapy-related clonal cytopenias of uncertain significance (t-CCUS). Complex karyotypes were found in four of seven patients who underwent karyotyping (57%). Next-generation sequencing identified TP53 mutations in 7 of 9 patients analyzed (78%). Among patients with germline testing, four (40%) did not have a germline mutation identified, four (40%) had a BRCA1 pathogenic/likely pathogenic (P/LP) variant, and two (20%) had a BRCA2 P/LP variant. Four patients received supportive care and/or observation after blood cancer diagnosis, and six received t-MN-directed therapy. The median survival for patients who received t-MN-directed treatment was 148 days. While cytopenias, particularly anemia, are known to occur with PARP inhibitor therapy, a subset of patients develop chronic cytopenias requiring bone marrow biopsy to evaluate for t-MN. Our study informs the expected findings of such biopsies.

The spatial anatomy of hematopoiesis in bone marrow has been extensively studied in mice and other preclinical models, but technical challenges have precluded a commensurate exploration in humans. Institutional pathology archives contain thousands of paraffinized bone marrow core biopsy tissue specimens, providing a rich resource for studying the intact human bone marrow topography in a variety of physiologic states. Thus, we developed an end-to-end pipeline involving multiparameter whole tissue staining, in situ imaging at single-cell resolution, and artificial intelligence (AI)-based digital image analysis, and then applied it to a cohort of disease-free samples to survey alterations in the hematopoietic topography associated with aging. Our data indicate heterogeneity in marrow adipose tissue (MAT) content within each age group, and an inverse correlation between MAT content and proportions of early myeloid and erythroid precursors, irrespective of age. We identify consistent endosteal and perivascular positioning of hematopoietic stem and progenitor cells (HSPCs) with medullary localization of more differentiated elements and, importantly, uncover new evidence of aging-associated changes in cellular and vascular morphologies, microarchitectural alterations suggestive of inflammaging, and diminution of a potentially active megakaryocytic niche. Overall, our findings suggest that there is topographic remodeling of human hematopoiesis associated with aging. More generally, we demonstrate the potential to deeply unravel the spatial biology of normal and pathologic human bone marrow states using intact archival tissue specimens.

ImportanceHereditary hematopoietic malignancies (HHMs) are hereditary cancer syndromes that constitute at least 14% of all myeloid malignancies, but genetic assays used to diagnose HHMs have historically been of variable quality. Here, we demonstrate that HHM assays continue to have persistent shortcomings. These diagnostic gaps place patients with HHMs at high risk for missed diagnoses, missed opportunities for cancer screening, and donor-derived leukemias following stem cell transplant. ObjectiveTo determine if the quality of HHM diagnostic assays has improved since 2020, when our group first demonstrated that most HHM diagnostic tests were insufficient for the accurate diagnosis of these syndromes. We hypothesized that the number of genes tested on each HHM assay increased from 2020 to 2022, in keeping with a more comprehensive sequencing approach. Design, Setting, and ParticipantsWe analyzed assays from eight commercial laboratories to determine which HHM-related genes were sequenced by these assays. We compared these assays to panels from 2020 to determine trends in sequencing quality. ResultsThe majority of HHM diagnostic assays did not change over time and are insensitive for the detection of the full spectrum of HHM-related mutations. The majority (75%) of HHM assays do not sequence CHEK2, the gene most frequently mutated in HHMs, and 25% of HHM assays do not sequence DDX41, the second most frequently mutated HHM gene. ConclusionsThe quality of HHM diagnostic assays has stagnated despite the discovery of novel HHM-related genes as well as prior work demonstrating heterogeneity in quality of HHM testing. The majority of commercially available HHM tests remain insufficient for the diagnosis of the full spectrum of HHM-related germline mutations. Key PointsO_ST_ABSQuestionC_ST_ABSHow have diagnostic assays for hereditary hematopoietic malignancies (HHMs) changed since 2020, when most HHM diagnostic assays were inadequate for the accurate diagnosis of HHMs? FindingsMost HHM assays have significant deficiencies in quality and do not sequence the most relevant HHM-related genes. No meaningful improvements in the quality of HHM diagnostic testing have occurred since 2020. MeaningThe quality of HHM diagnostic testing must be improved to universally include the most common HHM-related germline mutations. This will reduce the risk for false negatives, donor derived leukemias, improve genetic counseling, and improve screening for other HHM-related malignancies.

PurposeEvaluating the safety and tolerability of 5-FU-miR-15a (CR-001) in adults with relapsed or refractory acute myeloid leukemia (R/R AML) in a phase I dose-escalation study (EudraCT number: 2021-006332-46). Patients and MethodsA standard 3+3 dose-escalation study design was employed. Patients received intravenous (IV) administrations of the synthetic double-stranded mimic of miR-15a (miRNA) CR-001 of 7.5 mg, 11.25 mg, 15 mg and 18.75 mg/administration, weekly dosing. Each patient was dosed on a single dose level. The formulation included a fixed dose of GMP-grade linear polyethyleneimine (PEI; -Methyl-{omega}-hydroxy-poly[(iminioethylene)chloride]; Poly[imino(1,2-ethanediyl)]; CAS Number 26913-06-4; EC Number 608-018-6) as the excipient: 15 mg of PEI with 7.5 mg CR-001, and 20 mg of PEI with all higher dose levels. Glucose was added to stabilize the miRNA/PEI complexes. Biomarker analysis was performed using mass cytometry (CyTOF) on peripheral blood and bone marrow samples to quantify surface and intracellular protein expression at the single-cell level. ResultsA total of 11 patients with R/R AML (median age: 74 years; all classified as adverse risk per ELN 2022) received a cumulative 66 intravenous doses of CR-001 (range: 1-16 doses; median: 5) across the four dose levels. The treatment was well-tolerated. One patient demonstrated a partial clinical response, with resolution of extramedullary pericardial AML after four doses. Among the 10 evaluable patients, six achieved stable disease (SD) according to ELN criteria after four infusions. Single-cell profiling mass cytometry revealed molecular activity of CR-001 on known targets of miR-15a (e.g., BMI1, WEE1 and MCL-1) beginning at the lowest dose level (7.5 mg). Concurrently, an increase in BAX, a pro-apoptotic marker, was observed following treatment. ConclusionsCR-001 was well tolerated and demonstrated biological activity as evidenced by target modulation and disease stabilization in R/R AML patients. Biological activity was observed starting at the lowest dose level (7.5 mg/administration), with initial signs of clinical efficacy at 11.25 mg per administration. This trial was closed prematurely due to funding constraints, however, based on these findings further development is warranted, possibly in therapy combinations.

HighlightsO_LISingle-cell RNA sequencing of bone marrow from TP53-mutated AML patients showed a decrease in cells expressing anti-apoptotic genes like BCL2 and MCL1 after venetoclax and azacitidine treatment. C_LIO_LIImmune cells increased both in number and in gene expression levels associated with cytotoxicity post venetoclax combination therapy, verifying immune recovery. C_LIO_LIResidual AML cells expressed CD47 and CLL1, suggesting a role for ancillary treatment targeting antigens expressed on residual TP53-mutated AML cells. C_LI The BCL-2 inhibitor venetoclax combined with the hypomethylating agent azacitidine or with low-dose cytarabine significantly improves response rates and overall survival (OS) for newly diagnosed unfit and relapsed / refractory (R/R) acute myeloid leukemia (AML) patients. We retrospectively analyzed our experience with venetoclax combination therapy in 41 unfit AML patients (23 untreated, 18 R/R). Overall response rates were 78.3% for untreated patients and 61.1% for R/R patients. TP53 alterations were observed in 13 patients (31.7%) and were identified as an independent predictor of poor outcome (p=0.0008). We further conducted single-cell RNA sequencing in bone marrow, sampled before and after venetoclax and azacitidine treatment, of three TP53-mutated AML patients who achieved complete remission (CR) or CR with incomplete hematologic recovery. After treatment, numbers of cells expressing anti-apoptotic genes such as BCL2 and MCL1 decreased. CD4 T cells, cytotoxic CD8 T cells, and NK cells significantly increased both in number and in levels of gene expression associated with cytotoxicity post-treatment, confirming immune recovery in the tumor microenvironment. Residual AML cells expressed CD47 and CLEC12A (CLL1). These results indicate that venetoclax combination therapy of AML is promising in real-world clinical practice and suggest a role for ancillary treatment targeting antigens expressed on residual AML cells as a therapeutic strategy in TP53-mutated AML.