Blood Advances

Clonal hematopoiesis (CH) can predict treatment outcomes in solid cancers, but the prevalence and clinical impact of CH in previously untreated, aggressive large B-cell lymphomas (LBCL) remains poorly characterized. Moreover, CH may be detected in cell-free DNA (cfDNA)-based biomarker assays, but the longitudinal dynamics of CH mutations during and after immunochemotherapy in LBCL remain unknown. Hence, we characterized the spectrum of CH mutations in 247 patients with high-risk primary LBCL from three phase II clinical trials. We identified CH mutations in 23% of patients. Compared with healthy controls, TET2 mutations were enriched in peripheral blood (PB) samples of LBCL patients (P=0.01). CH clone size showed high concordance between PB and cfDNA (R2=0.59, P<0.0001) but was not associated with circulating tumor DNA (ctDNA) concentration (P-values 0.25-0.86). In contrast with prior reports in the context of recurrent lymphoma, CH mutation status was not associated with overall survival (OS) (P=0.78) or progression-free survival (PFS) (P=0.89) in our primary LBCL cohort. CH clones showed variable longitudinal dynamics during immunochemotherapy, with 18% of evaluable patients developing mutant CH clones, mostly with TP53/PPM1D mutations. Together, CH mutations can be readily detected in cfDNA of lymphoma patients, but they do not predict survival in primary high-risk LBCL.

Background: The ability to repopulate all lineages of blood cells from a single hematopoietic stem cell (HSC) requires a constant balancing between maintenance of quiescence/self-renewal and the need for differentiation and adoption of specific lineage phenotypes. A classical interpretation suggests that during homeostasis, long- and then short-term HSCs differentiate into multipotent progenitors (MPPs), which then lack self-renewal capacity but can still differentiate into all downstream lineages. While much is known about the intrinsic and extrinsic regulators that promote HSC homeostasis, less is known about factors which influence HSC behavior following significant bone marrow (BM) injury, such as sublethal irradiation (SLI). Methods: Conditional knock-out of follistatin-like 1 (FSTL-1) was achieved by breeding mice with floxed Fstl1 sites to mice expressing CreERT2 under control of the ROSA locus, followed by systemic tamoxifen administration. ELISA and RT-PCR quantitated protein and RNA level loss of FSTL-1, respectively. HSC populations were assessed by flow cytometry under multiple conditions. Sublethal irradiation induced BM injury and blocking polyclonal FSTL-1 antibodies were used to recapitulate results seen with genetic ablation. Finally, single cell RNA sequencing defined both cell-specific expression of Fstl1 within the BM compartment, but also identified transcripts and pathways upregulated in the absence of FSTL-1. Results: These studies delineate a novel role for FSTL-1 in regulating HSC biology. In addition to selective expression of Fstl1 within HSCs, genetic deletion of Fstl1 increases Lineage negative;Sca-1+c-Kit+ (LSK) cell number and alters HSC homeostasis. These findings are further amplified in the BM in the context of recovery from sublethal irradiation. Importantly, the post-irradiation increase in BM cellularity seen with Fstl1 deletion is replicated by treatment with anti-FSTL-1 polyclonal antibodies. Finally, single cell RNA sequencing reveals distinct Fstl1 expression in specific LSK subpopulations, including HSCs and proliferating cells, and identifies a novel relationship between decreased FSTL-1 levels and enrichment of canonical metabolic pathways including oxidative phosphorylation, fatty acid metabolism, and glycolysis. Conclusions: Taken together, these results highlight a novel role for FSTL-1 in promoting HSC homeostasis. They also unlock an innovative way to promote HSPC recovery following BM injury, knowledge predicted to be of significant therapeutic value.

Embryonic hematopoiesis is essential for establishing lifelong blood and immune system function. During development, hematopoietic stem and progenitor cells (HSPCs) acquire intrinsic programs that persist into adulthood and can influence disease susceptibility, yet the molecular signals governing these early-life decisions remain poorly understood. Here, we investigated the role of developmental Transforming Growth Factor-{beta} (TGF-{beta}) signaling in regulating HSPC lineage bias and long-term hematopoietic outcomes. Using the zebrafish model, we found that transient embryonic TGF-{beta} signaling inhibition during the HSPC specification window altered their frequency and migration after emergence from the hemogenic endothelium and movement into the key secondary maturation and expansion niche. Single-cell transcriptomic analysis of embryonic HSPCs revealed repression of migration- and cytoskeleton-associated genes alongside dampened expression of myeloid/macrophage-related genes following ALK5 inhibition. Functionally, early ALK5 blockade reduced macrophage numbers and promoted an M2-like immunosuppressive transcriptional profile. These developmental perturbations produced sustained effects on hematopoietic and immune function into adulthood, including diminished inflammatory gene expression, reduced clonal complexity, and impaired regenerative capacity. Together, our findings identify embryonic TGF-{beta} signaling as a key developmental regulator of HSPC fate and immune programming, with potential implications for immune dysfunction and susceptibility to inflammatory-related disease later in life. PAPER HIGHLIGHTS- Transient developmental signaling perturbations reshape hematopoietic trajectories - Embryogenic TGF-{beta} signaling instructs HSPC lineage priming and macrophage specialization - Early HSPC programming establishes persistent inflammatory states, clonal diversity, and modifies regenerative capacity

Mantle cell lymphoma (MCL) is a B-cell non-Hodgkin lymphoma characterized by heterogeneous clinical courses despite a common pathobiological initiating event. In this work we explore the genomic variants that characterize MCL and integrate transcriptomic data to comprehensively describe MCL biology. We performed whole exome sequencing (WES) on 28 tumor-normal pairs (lymph node and skin, respectively), as well as whole genome sequencing (WGS) and RNA sequencing on subsets of samples. We used established DNA and RNA analysis pipelines to detect single-nucleotide variants (SNV) and indels, structural variants, copy-number alterations, and RNA fusions. The canonical t(11;14)(q13;q32) CCND1::IGH translocation was detected in 8 of 10 WGS samples. Structural variant analysis additionally identified recurrent rearrangements involving KMT2A and PAFAH1B2. SNV and indel analyses revealed frequent mutations in ATM, TP53, CCND1, IGH, and NOTCH1. ATM exhibited diverse variant classes, including missense mutations, frameshift mutations, deletions, and duplications, while all detected NOTCH1 mutations were predicted loss-of-function frameshift variants. Copy-number analysis identified recurrent losses affecting DNA damage response genes, including TP53 and ATM, and recurrent gains involving transcriptional regulators and oncogenic signaling genes. Integrated pathway analysis demonstrated enrichment of transcriptional misregulation, DNA repair, PI3K/AKT signaling, and interleukin signaling pathways. We also identified recurrent alterations in candidate genes, including ASXL1, suggesting additional mechanisms of epigenetic dysregulation in MCL. Together, these findings provide a comprehensive description of somatic alterations in MCL and demonstrate that diverse genomic lesions converge on common pathways involved in genomic instability, transcriptional regulation, and tumor survival.

Haplo-identical hematopoietic cell transplantation (haploHCT) is an integral treatment paradigm for patients with leukemia. While overall survival (OS) post-haploHCT has steadily improved, relapse-free survival (RFS) remains relatively stagnant. Upon the discovery of killer immunoglobulin-like receptors (KIRs) on natural killer (NK) cells and their cognate human leukocyte antigen (HLA) ligands, algorithms have been developed to enhance graft versus leukemia effects. However, these algorithms fail to yield consistent predictions in patient outcomes. We utilized a combination of in silico protein folding and interactions to determine KIR:HLA reactivity in conjunction with in vitro acoustic force microscopy to measure cell avidity (CA) as a readout for KIR signal strength. CA was determined using monoallelic HLA expressing K562 cell lines, monoallelic KIR Jurkat cells, and peripheral blood NK cells. We extended the CA results and performed standard cytotoxicity assays as well. We discovered that HLA-B*35 interacts with KIR2DS4. We applied the newly discovered interaction to predict outcomes for HCT patients. Stratifying patients based on their HLA-B*35 positivity and donor KIR2DS4 status, we delineated a correlation to survival (P=0.061) when donors only had full-length KIR2DS4. Patients who received a haploHCT and NK cell addback from donors with only full-length KIR2DS4 had a significantly improved RFS (P=0.001) and OS (P=0.016) compared to truncated (KIR1D) and full-length KIR2DS4 donors. This was independently validated in a diverse 10/10 HLA matched European cohort with RFS (P=0.0255) and OS (P=0.0388). Thus, the identified novel KIR2DS4:HLA-B*35 interaction axis predicts patient survival, in both haplo-identical and fully matched, HCT and highlights that our current understanding of the KIR:HLA interactome is incomplete and requires remapping for enhanced therapeutic applications.

ERCC6L2 disease (ED) is an inherited bone marrow failure (BMF) syndrome that progresses almost exclusively to erythroid, TP53-mutated myeloid malignancy, but the somatic evolution underlying the progression in patients is unknown. We characterized the genetic landscape of 29 ED patients with longitudinal sampling, integrating whole-genome and single-cell RNA sequencing, and compared the findings with Shwachman-Diamond syndrome (SDS), another TP53 mutation-prone disease, to define somatic evolution across disease stages. ED was defined by early, intense, and recurrent selection of TP53-mutant clones: TP53 mutations were more frequent, more often multiple, and showed steeper age-related expansion than in SDS; all TP53-negative ED patients were children. The burden of multiple TP53 mutations exceeded that of sporadic TP53-mutated myeloid neoplasia and was evident before malignancy. Missense mutations dominated the BMF phase, whereas non-missense mutations were seen in patients with malignancy, consistent with biallelic second hits, marking a stage-dependent shift in p53 inactivation. Beyond TP53, the landscape was strikingly restricted, with PPM1D as the only recurrent driver gene. Notably, despite the DNA-repair function of ERCC6L2, no ED-specific mutational signature emerged; but instead reflected age-associated processes. Malignant progression was accompanied by a complex karyotype, recurrent chromosome 5, 7, and 12p losses enriched in erythroid progenitors, and kataegis, consistent with branched, erythroid-predominant clonal evolution. Our findings establish ED as a paradigm of preleukemic dependency on p53-pathway attenuation. As progression is driven predominantly by p53 and captured longitudinally from the earliest clones, ED offers a model of how TP53-mutated leukemia arises and evolves, with relevance beyond a rare syndrome.

Background/Objectives: Many myeloid malignancies are defined by recurrent structural variants (SVs) and copy-number alterations (CNAs) under the current World Health Organization (WHO) and International Consensus Classification (ICC) frameworks. Detecting these abnormalities typically requires several standard-of-care (SOC) assays, each with its own blind spots, and then laboratories and clinicians are left to piece the results together. Genome mapping has emerged as a mainstream technology with the potential to serve as a high-resolution alternative to several standard-of-care cytogenetic assays such as karyotyping (KT), fluorescence in situ hybridization (FISH) or chromosomal microarray (CMA). Our laboratory at Augusta University has published numerous publications on the analytical and clinical utility of optical genome mapping (OGM) as a first-line assay for hematological malignancies. Electronic genome mapping (EGM) pursues the same goal with an electronic rather than optical readout. The objective of this study was to demonstrate the performance and utility of EGM as a valuable tool to detect clinically relevant SVs and CNAs found by SOC testing. Cytogenomic characterization of hematologic malignancies commonly requires multiple complementary assays, including karyotyping, FISH, and chromosomal microarray, each with distinct analytical limitations. Electronic genome mapping (EGM) provides genome-wide interrogation of structural variants and copy-number alterations using electronic single-molecule detection. We evaluated the feasibility and analytical performance of EGM in previously characterized myeloid malignancies. Methods: EGM was performed on the Nabsys OhmX platform using five previously characterized bone marrow aspirates from cases with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML). EGM calls were compared with findings from karyotyping (KT), fluorescence in situ hybridization (FISH), and OGM. Results: All five samples passed quality control, with contig N50 values ranging from 1.8 to 3.8 Mb and more than 90% of reference labels represented in the assembled contigs. EGM detected every SOC-reported abnormality including two t(9;22)(q34.12;q11.23)/BCR::ABL1 rearrangements, trisomy 4, trisomy 21, and monosomy 7. In the most complex case, EGM resolved a del(20q) reported by KT and FISH into an intrachromosomal fusion, fus(20;20)(q11.21;q13.12), showing not only what was lost but where the chromosome rejoined. Like OGM, EGM identified an approximately 1.8 Mb loss at 7q22.1 involving 45 of genes of which only CUX1 is associated with cancer. This 7q loss was not detected by KT or FISH. Interestingly, EGM also detected the KMT2A-PTD variant previously detected by OGM. Collectively, EGM demonstrated 100% concordance (6/6 SOC-reported abnormalities; 9/9 OGM calls). Conclusions: In this pilot study on five hematological malignancy specimens, EGM matched SOC and OGM across translocations, aneuploidies, insertions and deletions, including BCR::ABL1 rearrangements in both AML and CML, and added high-resolution breakpoint detail beyond karyotype resolution. These results support larger studies across more subtypes to establish EGM as a first-line genome-wide cytogenomic tool for hematological malignancies. Keywords: Genome mapping, Electronic genome mapping, hematological malignancies, structural variants, copy number variants

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy associated with a poor prognosis and an unmet need for more effective treatment options, particularly after relapse. One potential approach to address this need, which has been used successfully in solid tumors, is adoptive transfer of T cells engineered to express specific T cell receptors (TCRs). Particularly attractive are TCRs that can target epitopes derived from oncogenes on which cancer cells depend while sparing normal cells that do not harbor the mutation. One promising oncogenic target in AML is mutated nucleophosmin 1 (mtNPM1), present in up to 30% of AML cases. Most NPM1 mutations result in a common frameshifted sequence that generates peptides presented by several class I human leukocyte antigens (HLAs), such as HLA-A*11:01. In this study, we vaccinated HLA-A*11:01-transgenic mice with the mtNPM1 peptide AVEEVSLRK and isolated six functional TCRs from mtNPM1-reactive lymphocytes. When engineered into human T cells, two of these TCRs consistently mediated robust and specific lysis of an AML cell line and primary AML cells co-expressing HLA-A*11:01 and mtNPM1. Neither TCR recognized related human peptides, HLA-A*11:01-expressing normal peripheral blood mononuclear cells or stem/progenitor cells, or cells expressing diverse allogeneic HLAs, suggesting a low risk of cross-reactivity and alloreactivity. In an AML xenograft model, both TCRs controlled AML in hematopoietic organs, although extramedullary tumors emerged during treatment, potentially reflecting the aggressive behavior of the model that may not be representative of AML in patients. Together, these data suggest that the two TCRs represent strong candidates for further preclinical development in adoptive T cell therapy for NPM1-mutated, HLA-A*11:01-positive AML. These results also support the use of HLA-transgenic mice as a platform for generating TCRs targeting oncogene-derived epitopes in hematologic malignancies.

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy characterized by excess inflammatory signaling and clonal myeloproliferation. The relative contribution of neutrophils (PMNs) to the inflammatory milieu in CMML is poorly understood. In this study we sought to understand whether neutrophil extracellular trap (NET) formation, a key mediator of neutrophilic inflammation, is dysregulated in CMML and can be therapeutically targeted with a novel peptide inhibitor of NETosis called neonatal NET-inhibitory factor (nNIF). Here, we demonstrate that baseline NET formation is aberrantly increased in primary CMML PMNs transcriptionally primed for NETosis, and that soluble factors produced during CMML NET formation promote clonogenicity in CMML CD34+ hematopoietic cells matched to the same patient. Further, we show that nNIF and clinical agents under investigation in CMML effectively inhibit NETosis, warranting further study of NET inhibitory agents in this rare disease with limited treatment options.

Large genomically annotated acute myeloid leukaemia (AML) datasets exist, but population-based contemporary cohorts remain scarce. Here we report clinicopathological, genomic, and outcome data from Danish AML patients. 2,512 AML patients were identified between 2015-2022, of whom 33.8% had available NGS data (NGS+). In patients [&le;]70 years, baseline characteristics and outcomes were comparable between NGS+ and NGS- groups. In patients >70 years, more NGS+ patients received intensive treatment, but survival was similar among intensively treated patients. The distribution of mutations varied significantly by age and sex, with older age and male sex exhibiting higher frequencies of adverse-risk gene mutations. In intensively treated NGS+ patients, ELN2017 stratified 5-year OS: 58.4% (favorable), 43.4% (intermediate), and 28.2% (adverse), with hazard ratios (HRs) of 0.63 (favorable) and 1.45 (adverse) relative to intermediate. ELN2022 yielded corresponding OS rates of 56.9%, 51.8%, and 29.7%, with HRs of 0.78 and 1.86. The two models had comparable predictive performance for OS in a time-dependent model. In conclusion, outcomes of intensively treated AML patients were comparable irrespective of NGS status, underscoring the representativeness of the REFORM-AML database for the Danish AML population. Age and male sex correlated with adverse-risk mutations, and both ELN2017 and ELN2022 robustly predicted survival.

Purpose: Adults with high- or very high-risk acute leukemia (AL) or myelodysplastic syndrome (MDS) face substantial relapse risk after allogeneic hematopoietic stem-cell transplantation. We evaluated single-unit cord blood (CB) transplantation after ex vivo expansion with UM171 in this population. Patients and Methods: Two prospective, single-arm phase II trials at four centers enrolled 64 adults with high- or very high-risk AL or MDS; 60 received a UM171-expanded CB transplant and comprised the analysis population. CB units were preferentially selected at a 5/8 HLA match to maximize the graft versus leukemia effect. Patients received intermediate- or high-intensity conditioning with tacrolimus/mycophenolate mofetil graft-versus-host-disease (GVHD) prophylaxis. Endpoints included safety, feasibility, non-relapse mortality (NRM), relapse-free survival (RFS), overall survival (OS), GVHD, GVHD-free relapse-free survival (GRFS), chronic GVHD-free relapse free survival (CRFS). Results: Thirty-two percent of patients had undergone previous transplantation, 17% of patients with AL were not in remission and 24% of those with AML/MDS had TP53 mutations. Of 62 patients who remained eligible for transplantation, 60 had a graft successfully manufactured and infused. Median times to neutrophil and platelet engraftment were 17 and 38 days, respectively. NRM was 5.1% at day 100 and 15.2% at 1 year. Two-year cumulative incidence of relapse was 22.3%. Two-year OS and RFS were 63.9% and 60.4%, respectively. Grade III-IV acute GVHD incidence was 20.3% at 1 year and moderate-to-severe chronic GVHD incidence was 6.8% at 2 years. Conclusion: UM171-expanded CB transplantation was feasible and provided prompt engraftment, durable disease control, and infrequent clinically significant chronic GVHD in adults with high- and very high-risk AL/MDS. Comparative studies are warranted to define its role relative to contemporary donor platforms.

Immunotherapy has transformed treatment for many cancers. In the aggressive and genetically heterogeneous diffuse large B-cell lymphoma (DLBCL), CD19 CAR T-cell therapy is highly effective, whereas immune checkpoint blockade has shown limited benefit. Loss of MHC expression is a common mechanism to escape T-cell cytotoxicity, and loss of MHC class I (MHC-I) and II are frequent in DLBCL. We applied imaging mass cytometry to diagnostic biopsies from younger, high-risk DLBCL patients to map the tumor microenvironment (TME) spatial architecture in relation to tumor cell MHC expression, mutational status, transcriptomic and proteomic profiles. Neighborhood analyses identified four TME subtypes: immune-cell depleted and three immune-infiltrated types (mixed, CD4 T cell-rich, CD8 T-cell/macrophage-rich). Depleted cases had shorter overall survival (p = 0.033) and increased expression of proteins involved in DNA replication and proliferation markers compared to infiltrated cases. Tumor cell MHC-I expression was heterogeneous. Cases with low frequency of MHC-I-pos tumor cells were enriched for the depleted TME type. MHC-I-pos tumor cells were surrounded by CD4 and CD8 T cells and M1 macrophages, whereas MHC-I-neg tumor cells were closer to other MHC-I-neg tumor cells. These findings suggest that TME-based classification incorporating tumor cell MHC-I status may improve individualized immunotherapy selection.

The adult hematopoietic system is established during embryogenesis through the generation of hematopoietic stem and progenitor cells (HSPCs) within specialized transient niches, yet the signaling mechanisms that coordinate this process remain poorly understood. Here, we identify a developmental role for kainate receptors (KARs), glutamate-gated ion channels classically associated with excitatory neurotransmission, in coordinating primitive hematopoiesis and hemogenic niche formation in zebrafish. Loss of the KAR subunits encoded by grik1b or grik5 depletes primitive blood cells and disrupts hemogenic niche organization. These defects are accompanied by endothelial oxidative stress and impaired Notch signaling, which together reduce HSPC emergence and limit the clonal diversity of the adult hematopoietic stem cell pool. Shifting primitive hematopoietic output away from erythropoiesis and toward myelopoiesis restored macrophage niche colonization, endothelial Notch activity, and HSPC formation in KAR-deficient embryos. Moreover, hemogenic cell-specific Notch reactivation rescued HSPC production, demonstrating that KAR-dependent primitive hematopoiesis regulates this process through endothelial Notch signaling. Lastly, conserved KAR expression in endothelial and hematopoietic populations across zebrafish, mouse, and human suggests that these receptors may regulate blood development across multiple cellular components. Together, these findings reveal that glutamate receptor signaling extends beyond its well-established functions in neuronal transmission to coordinate the developmental processes that establish the blood system.

Diffuse B-cell lymphomas are the most prevalent canine hematologic malignancies, and their clinical presentation resembles that of human B-cell non-Hodgkin lymphomas (NHLs). However, a lack of a clear subtyping framework perpetuates imprecise treatment approaches that fail to address mechanisms of therapy resistance. Here, we employed deep phenotyping via serial sampling and longitudinal transcriptomics to evaluate intratumoral composition and therapy response in a 6-year-old neutered male goldendoodle with stage 5A marginal zone lymphoma (MZL). Following sequential treatment consisting of a single IV dose of oncolytic vesicular stomatitis virotherapy (VSV) and standard CHOP chemotherapy 30 days later, we performed scRNAseq on seven serial lymph node biopsies and spatial sequencing on a resection taken 10 days post-VSV treatment. B cells (>90%) comprised three transcriptionally distinct and temporally stable subpopulations, with specific copy number profiles and robust spatial organization despite disrupted lymph node architecture. Analysis across subpopulations revealed downregulation of the quiescence program including KLF2, suggesting similarity to KLF2-deficient human MZL. While VSV successfully reached target B cells, downstream transcriptional responses were predominantly localized to the T-cell compartment. T cells ([&le;]6%) showed increased proliferation and upregulation of cytotoxicity markers post-VSV administration, enriching for leukocyte-mediated cytotoxicity pathways consistent with an anti-viral immune response. Ultimately, these results provide an in vivo proof-of-concept for the treatment paradigm in canine lymphoma, while emphasizing the need for improved subtyping frameworks and multidimensional treatment strategies targeting intratumoral heterogeneity.

Background: Relapse remains the principal cause of treatment failure in pediatric acute leukemia. Clinically validated minimal residual disease (MRD) assays - multiparameter flow cytometry, immunoglobulin/T-cell-receptor rearrangement sequencing, and targeted next-generation sequencing panels -- are highly sensitive but read out predefined features and do not reconstruct clonal architecture or link a cell's genotype to its functional state. We systematically reviewed what single-cell sequencing has revealed about clonal evolution, chemoresistance, the immune microenvironment, and post-therapeutic immune dynamics in this disease. Methods: This review followed PRISMA 2020. PubMed/MEDLINE, the Cochrane Library, and Google Scholar were searched to September 2026 for primary studies applying single-cell DNA, RNA, chromatin-accessibility, surface-protein, or immune-repertoire sequencing to pediatric acute leukemia samples or pediatric-derived models. Risk of bias in non-randomized studies was assessed with ROBINS-I. Because designs, platforms, and outcomes were heterogeneous, findings were synthesized narratively by biologic theme without meta-analysis, following the SWiM guideline. Results: Of 63 records identified, 10 studies were included (published 2016-2026; 6-159 patients or samples per study; up to ~700,000 cells profiled). Single-cell DNA sequencing traced minor NOTCH1-mutant diagnostic subclones into PTEN- or TP53-mutant relapse clones that bulk sequencing missed. Rare dormant, label-retaining cells and developmental-state-dependent drug sensitivity defined chemoresistant compartments. Remodeling of the bone marrow immune microenvironment identified patients with adverse outcome among those classified as MRD-low, and pre-infusion CAR T-cell type 2 functionality and post-transplant cytotoxic memory CD8+ T-cell subsets were associated with durable remission. Of the seven studies assessed with ROBINS-I, four were at moderate and three at serious overall risk of bias, driven chiefly by small, selected cohorts. Conclusions: Single-cell sequencing yields mechanistic insight that complements, rather than replaces, clinically validated MRD assays, and nominates candidate biomarkers for risk stratification, treatment selection, cell-therapy manufacturing, and post-transplant monitoring. The evidence remains preliminary; adequately powered, prospective, and harmonized studies are required before clinical adoption.

IDH inhibitors promote differentiation and hematological improvement in myeloid malignancies by reversing epigenetic dysregulation. However, molecular predictors of response remain poorly defined. We retrospectively analyzed 79 patients with IDH mutated myeloid malignancies treated with either ivosidenib (IDH1; n=36) or enasidenib (IDH2; n=43). Hematologic benefit was assessed using a composite complete hematologic response (CHR), defined by neutrophil and platelet recovery, and transfusion independence. Targeted sequencing was performed at baseline and at best hematologic response. Associations between co mutations, hematologic response, and treatment duration were evaluated using multivariable models. Overall, 43% of patients achieved CHR, with similar rates between inhibitors. Ivosidenib treated patients achieving CHR demonstrated a significant reduction in IDH1 VAF (p=0.010), which was not observed in enasidenib-treated CHR patients. Within the CHR cohort, ivosidenib patients had a greater reduction in IDH VAF relative to enasidenib patients (mean VAF change -16.74% vs -0.05%, p=0.034). Ivosidenib responders demonstrated a trend toward more durable treatment duration compared with enasidenib responders (p = 0.0908). RUNX1 and BCOR mutations were significantly enriched among patients with incomplete hematologic response and were independently associated with shorter treatment duration after adjustment for clinical factors (RUNX1: HR 2.53, p=0.0024; BCOR: HR 2.31, p=0.0058). IDH1 inhibition was associated with clonal contraction and durable hematologic benefit, a pattern not observed with IDH2 inhibition. Co mutations in RUNX1 and BCOR identified patients unlikely to achieve sustained hematologic improvement, suggesting a secondary block to differentiation. These findings support the integration of mutational and molecular profiling to refine patient selection and guide combinatorial strategies in IDH mutated myeloid malignancies.

While professional antigen-presenting cells drive adaptive immunity, atypical cell types can fulfill this role in the bone marrow. Megakaryocytes (MKs) are canonically recognized for platelet production, but recent studies indicate functional heterogeneity and immune potential. We found that ~20% of bone marrow MKs express Major Histocompatibility Complex (MHC) II and co-stimulatory receptors CD80, CD86, CD40, and CD83. These MKs process and present antigen to activate T cells ex vivo in an MHC II-dependent manner. MK/T cell interactions induced TGF-{beta}1 secretion and promoted induced Treg differentiation. Prior stimulation of MKs with LPS or Poly I:C was associated with modest Th1-associated CD4+ T cell responses, including IFN-{gamma} and TNF- production, without robust Th17 differentiation. Immunopeptidomics of the murine MK MHC II receptor confirmed occupancy by exogenous peptides, suggesting in vivo functionality. Using a murine model with MK-targeted deletion of MHC II (Pf4-MHC{Delta}/{Delta}), we observed altered TLR signaling and reduced bone marrow TGF-{beta}1. Together, these findings identify MHC II+ MKs as noncanonical antigen-presenting cells with the potential to modulate CD4 T cell responses as part of the immune regulation of the bone marrow niche.

The human T cell repertoire is generated through recombination of a multitude of T cell receptor gene segments, yielding a complex array of T cell clones. T cell receptor beta (TRB) V gene segment defined, T cell clonal frequency when organized in correspondence with the respective V gene segment positions on the TRB genomic locus yields a periodic, undulating curve in the spatial domain. Using the genomic distance from the TRB-D1 segment to the TRB-V1-29 segments, spectral analysis was performed utilizing Lomb-Scargle periodogram (modified Fourier analysis) to obtain spectral power curves quantifying the TRB V clonal frequencies from 12 allogeneic stem cell transplant donors (baseline) and recipients (<100 days or b/w 100-365) using a variety of analytic software. Patients either underwent HLA matched related bone marrow or HLA matched unrelated blood stem cell transplantation, using PT-CY for the former and ATG for the latter. Spectral Power curves revealed dominant spectral peaks at wavelengths ranging from 113-335 millicycles/kb in the 12 donors, with consistent frequency domain spectral patterns, supporting similar use of V segments across healthy individuals. PT-CY recipients had power spectra closely corresponding to the respective donors, however the ATG recipients had relatively dispersed spectra, with spectral centroid shifted towards higher frequencies compared to donors and a reciprocal decline in the low frequency indices. Spectral power was concentrated in the <3 kb and 3-12 kb wavelengths in both groups, consistent with the periodicity observed in the relative V gene segment usage across the repertoire. The analyses reported here demonstrate that the healthy SCT donors have a spectral signature occupying short to intermediate wavelegnths in the frequency domain, PT CY recipients recover with donor-like spectra on the average, however ATG recipients tend to shift towards higher frequencies. These findings are consistent with a normal organized distribution of TRB V segment usage in healthy individuals, with PT-CY preserving this organization more consistently than ATG. Spectral (Fourier) analysis of TRB sequencing data provides a repertoire wide summary of T cell clonal distribution.

Type 2B von Willebrand disease (VWD) is characterized by missense variants in exon 28 of the von Willebrand factor (VWF) gene that encodes the VWF-A1 domain. These missense mutations result in single amino acid residue substitutions that cause activation of VWF-A1 and promote spontaneous interaction with platelet GPIba;. Importantly, enhanced VWF clearance has been shown to play a key pathogenic role in >90% patients with Type 2B VWD. Although this VWF clearance occurs via mechanisms that are independent of VWF-platelet complex formation, the biological mechanisms responsible for the increased clearance of type 2B VWD variants remain poorly understood. In this study, we investigated a series of different type 2B amino acid substitutions within the VWF-A1 domain (R1306W, R1308C, W1313C and R1379L). We demonstrate that these variants all exhibit significantly increased macrophage binding. In part, the enhanced macrophage interactions are mediated via increased binding of type 2B VWD variants to LRP1 extracellular cluster II and IV. Our findings further demonstrate that the K1405-K1408 lysine cluster in the VWF-A1 domain plays a key role in enabling enhanced LRP1 interactions for type 2B VWD variants. In addition, we show that type 2B VWF variants demonstrate significantly enhanced interaction with the macrophage MGL receptor. Finally, and importantly from a clinical perspective, we demonstrate that the increase in macrophage binding for type 2B variants is significantly attenuated in the presence of BT200. Collectively, our findings have direct translational relevance with respect to the clinical heterogeneity and treatment of type 2B VWD.

Background: The clinical course of acute leukemias and related hematologic neoplasms is defined by aggressive clonal proliferation, severe hematopoietic disruption, and recurrent molecular alterations. Although serial bone marrow biopsies, multiparameter flow cytometry, and next-generation sequencing remain standard surveillance modalities, procedural invasiveness, high costs, and operational turnaround times limit their frequency. Minimally invasive, scalable biomarkers integrating routine hematologic indices, targeted gene expression, circulating oncoprotein levels, and patient compliance metrics may provide an accessible framework for dynamic monitoring. Methods: In this longitudinal cohort study of 66 patients across 272 serial encounters (discrete follow-up intervals: days 0, 14, 41, 71, and 78), we assessed the prognostic value of peripheral complete blood counts (white blood cell count [WBC], hemoglobin [Hb]), molecular indicators (FLT3, NPM1, and DNMT3A transcript levels; targeted soluble FLT3 quantification), and medication adherence. The primary clinical endpoint was longitudinal progression to critical deterioration or disease-related mortality. Generalized linear mixed-effects models (GLMM) with patient-specific random intercepts were constructed to account for intra-subject repeated measures. Predictors were standardized per 1-SD increment. Model performance was assessed via the Akaike Information Criterion (AIC) and the Concordance index (C-index). Results: Leukocyte expansion correlated inversely with hemoglobin concentration (r=-0.877, p<0.001), reflecting bone marrow infiltration and secondary erythroid suppression. Multivariable mixed-effects modeling confirmed that elevated WBC count (adjusted odds ratio [aOR] = 1.52 per 1-SD increase; 95% CI: 1.24-1.86; p<0.001) and reduced medication adherence (aOR = 0.58; 95% CI: 0.44-0.76; p<0.001) independently predicted adverse clinical transition. Integrating targeted molecular markers (FLT3 expression and soluble protein levels) with routine hematologic indices and adherence demonstrated superior calibration and discrimination over single-domain models ({Delta}AIC[&ge;]116; Harrells C-index = 0.81). Conclusions: Readily accessible peripheral hematologic indices, targeted molecular quantification, and medication adherence provide complementary prognostic information regarding disease activity. Integrating these low-burden parameters into clinical workflows offers an efficient, scalable paradigm for early risk stratification in hematologic malignancies.