Blood Cancer Journal

PURPOSEBispecific antibodies (BsAbs) represent a major advancement in the management of relapsed/refractory multiple myeloma (RRMM), offering high response rates even in heavily pretreated patients. However, their use presents operational, safety, and supportive care complexities that require coordinated care teams, and evolving infrastructure. This manuscript summarizes best practice recommendations for adverse event (AE) management, outpatient operational models, referral pathways, and emerging strategies to optimize long-term tolerability. METHODSMedlive--A PlatformQ Health Brand conducted qualitative interviews of academic and community-based clinicians. Discussions focused on BsAb implementation, patient selection and counseling, and AE management. Experts provided recommendations on team-based protocols, transitions of care, and inpatient versus outpatient considerations. RESULTSTen hematologists/oncologists (academic n=4; community n=6) described practice patterns, barriers, and perspectives on BsAb use. BsAbs were consistently regarded as highly effective across multiple lines of therapy, particularly for patients without alternatives. Cytokine release syndrome (CRS) was the most common acute toxicity, generally low grade and managed effectively with early tocilizumab, including prophylactic use in outpatient settings. Immune effector cell-associated neurotoxicity syndrome (ICANS) was rare, mild, and best mitigated through early recognition and caregiver support. Infections, largely from BCMA-associated hypogammaglobulinemia, frequently interrupted therapy, necessitating antiviral prophylaxis, pneumocystis jirovecii pneumonia (PJP) prophylaxis, and intravenous immunoglobulin (IVIG). Outpatient step-up dosing is expanding, supported by prophylactic strategies and academic-community collaboration. Timely referral was emphasized to preserving eligibility. Major outpatient challenges included sequencing, infrastructure readiness, and standardized caregiver and staff education. CONCLUSIONEffective community implementation of BsAbs requires multidisciplinary coordination, standardized AE protocols, infection prevention, and infrastructure to support monitoring, referrals, and equitable access. These measures are critical to ensure safe, sustainable integration of bispecific therapies and to optimize patient outcomes.

BackgroundChimeric antigen receptor (CAR) T cell therapy has transformed the treatment of B cell malignancies, but translation to acute myeloid leukemia (AML) has been hindered by on-target, off-tumor (OTOT) toxicity. In particular, endothelial cell (EC)-specific toxicity has limited clinical translation of promising leukemia stem cell-enriched targets such as CD93. Innovative strategies to mitigate EC damage while preserving antileukemic efficacy are needed. MethodsWe hypothesized that a NOT-gated CAR T cell strategy could circumvent EC toxicity associated with CD93 targeting. Considering CAR target antigen density and the pro-inflammatory microenvironment of CAR T cells, we identified VE-cadherin (VC), a highly specific EC marker, as an optimal inhibitory CAR target. We engineered a novel VC-specific single chain variable fragment (scFv), confirmed EC specificity in the context of a VC-specific second-generation activating CAR, then evaluated VC/CD93 NOT-gated CAR T cells for EC protection and antileukemic activity in in vitro cytotoxicity assays and in a three-dimensional vascularized microphysiological system. ResultsVC/CD93 NOT-gated CAR T cells maintain potent cytotoxicity against AML across multiple effector-to-target ratios, but preserve EC integrity, including in a three-dimensional vascular model system. Importantly, prior AML exposure did not impair the EC-protective function of the VC-specific iCAR, indicating durable NOT-gate activity under inflammatory conditions. Conversely, EC-induced iCAR inhibitory functions did not limit downstream antileukemic cytotoxicity, confirming a reversibility of both activation and inhibitory signals. Conclusions: These findings establish NOT-gated CAR T cells as an effective strategy to overcome EC-specific OTOT toxicity. Our results underscore the importance of CAR target discovery and validation across a spectrum of inflammatory states that can influence antigen expression and available therapeutic windows. This approach expands the potential CAR target landscape for AML and may be more broadly applicable to other malignancies where OTOT toxicity limits clinical translation.

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy. Key PointsO_LIRAS mutations independently predict unfavorable survival after CAR-T CD19 in pediatric B-ALL. C_LIO_LIRAS mutations increase risk of CD19 negative relapse after CAR-T CD19 therapy in pediatric B-ALL. C_LI

Background: Modern therapy for childhood and adolescent leukemia requires accurate risk classification of genomic subtype. Although short-read next-generation sequencing (NGS)- based approaches provide comprehensive clinical diagnostics in limited, highly resourced settings, they remain expensive, slow, and inaccessible to most children worldwide. Transformative approaches are needed to improve diagnostic classification for leukemia globally. Methods: We simultaneously continued to develop an analytical pipeline NASVar (Nanopore variant calling for adaptive sampling), and conducted a multicenter, type-two hybrid clinical validation study of an Oxford Nanopore Technologies (ONT) adaptive-sampling whole-genome sequencing (asWGS) assay across hospitals with varying diagnostic resources. In preparation for implementation, a global panel developed a leukemia-based standardized gene set and consensus laboratory-developed test (LDT) validation guidelines. Measures of assay effectiveness compared to both conventional and orthogonal NGS methods, where available, were simultaneously collected with data to measure the implementation outcomes of feasibility, fidelity, appropriateness, and cost. Results: All four centers successfully completed the LDT validation, with minimal adaptations required for regulatory compliance. A total of 457 specimens were sequenced (331 B-ALL, 83 AML, 43 T-ALL). For the 210 B-ALL cases with locally resolved genomic subtypes defined by DNA alterations, asWGS was 100% concordant (210/210). Cases locally defined as B-other were resolved via asWGS with disease-defining DNA alterations in 47% (49/105) of cases. An additional 41% (43/105) of locally defined B-other cases were classified by incorporation of DNA methylation, and all 16 B-ALL patient-derived xenograft controls were correct, for a total of 96% (318/331) of all B-ALL cases in the cohort resolved with single assay asWGS. For AML, 97% (56/58) of cases with locally resolved genomic subtypes were identified by automated asWGS analysis, while an additional two cases were identified after targeted manual review. At Indus Hospital in Pakistan, the B-ALL and AML diagnostic genomic subtype yield increased from 28% with local standard of care diagnostic testing, to 84% with asWGS. The cost of reagents and consumables in the United States, assuming pooled three-plexing, was $343/sample. Based on the combined hybrid validation results, all centers are independently preparing for clinical return of results. Conclusions: ONT asWGS was successfully validated as a clinical assay in four diverse hospital settings. As a single, multi-omic platform that delivers value across the continuum of high-resource to resource-limited contexts, the approach offers a disruptive solution to address the global equity gap in cancer diagnostics.

Long-term follow-up of the CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with daratumumab, both in combination with bortezomib, thalidomide, and dexamethasone during induction and consolidation, and during maintenance therapy, in transplant- eligible patients newly diagnosed with multiple myeloma (MM). However, outcomes among CASSIOPEIA patients remain heterogeneous across treatment groups. Measurable residual disease (MRD) is a strong indicator of the depth and duration of therapeutic response and is independently associated with both PFS and overall survival (OS), but it does not fully capture the biological diversity of MM. We performed a risk prediction analysis based on transcriptomic subgroups in CASSIOPEIA patients. A subset of 628 patients was characterized using RNA sequencing and consensus clustering identified five transcriptomic subtypes of MM. Long-term follow-up allowed the definition of three transcriptomic risk categories, with estimated 72-month PFS rates of 70%, 51%, and 27% for low, intermediate, and high-risk groups, respectively, among patients who received daratumumab in at least one treatment phase. In these patients, MRD negativity rates after consolidation and six months later were significantly higher in the low and high-risk groups compared with the intermediate-risk group. In the high-risk group, MRD status was not associated with PFS or OS. This suggests that, although daratumumab administered during both the induction/consolidation and maintenance phases improves the clinical outcomes of patients with activation of NSD2 or overexpressing members of the MAF family, highly aggressive minor clones may rapidly expand. These findings emphasize the need for novel therapeutic strategies in this high-risk population.

Chimeric antigen receptor (CAR) T-cell therapy has transformed treatment for relapsed /refractory(r/r) lymphoid malignancies. Yet, these cellular immunotherapies are often associated with immune-related adverse events (irAEs), namely cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), that pose significant risks to patient safety and limit broader clinical implementation of CAR T-cell therapies. In the current study, we used proteomics technology to establish circulating protein signatures that would predict severe CRS and ICANS in r/r lymphoma patients that subsequently received CAR T-cell therapy. Initial discovery was performed using plasma samples collected preceding CAR T-cell infusion from 39 r/r lymphoma patients at MD Anderson Cancer Center. A 5-marker and 8-marker protein panel was developed for predicting Grade [&ge;] 2 CRS and ICANS respectively, yielding respective AUCs of 0.85 [95% CI: 0.72-0.98] and 0.91 [95% CI: 0.81-1.00]. Independent testing of the CRS and ICANS panel was performed in a cohort of 59 r/r lymphoma patients from the Moffitt Cancer Center, with resultant AUCs of 0.76 [95% CI: 0.63-0.89] and 0.67 [95% CI: 0.51-0.84] for the CRS and ICANS panel, respectively. Patients were further classified into low-, intermediate-, and high-risk groups based on panel score tertiles. In the combined dataset (MDACC + Moffitt), compared to patients in the low-risk group (reference), patients in the intermediate- and high-risk groups were 3.15 [95% CI: 0.92-12.71] and 13.84 [95% CI: 4.21-56.26] more likely to have Grade [&ge;] 2 CRS, and 1.21 [95% CI: 0.36-4.23] and 8.59 [95% CI: 2.87-29.09] more likely to have Grade [&ge;]2 ICANS. The protein biomarker panels provide a means to risk stratify patients who are at high risk for developing severe CRS and ICANS, to inform on the need for prophylactic interventions and improve patient outcomes.

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm with very heterogeneous clinical and biological behavior. Among molecular variables, TP53 alterations are well-established adverse prognostic markers; however, MYC activation, which has been linked to disease progression, has not been completely defined in terms of clinical and biological impact, particularly in relation to TP53 status. Here, we investigated the effects of MYC overexpression according to TP53 status using clinical and transcriptomic data from CLL patients and novel cellular models. CLL patients with TP53WT and MYC overexpression exhibited significantly shorter time to first treatment and overall survival, indicating an aggressive disease course comparable to that of patients with TP53 alterations. Consistently, MYC overexpression in in vitro TP53WTmodels was associated with increased proliferation, enrichment of AKT/mTOR signaling and upregulation of genes involved in leukemogenesis and tumor progression such as FOXO6. Moreover, MYC overexpression was associated with increased sensitivity to venetoclax in TP53WT cells. By contrast, the concurrence of MYC overexpression and TP53 dysfunction conferred resistance to conventional CLL therapies such as BCL2 or BTK inhibitors. Of note, we identified a glycolysis inhibitor, in monotherapy or combined with BKT inhibitors, as a potential therapeutic strategy for CLL patients harboring MYC overexpression and TP53 alterations.

Richter transformation of Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) into classic Hodgkin lymphoma (CHL-RT) is rare and remains incompletely understood. Two histologic subtypes are recognized: type 1 (CLL/SLL with scattered Hodgkin/Reed-Sternberg (HRS) cells) and type 2 (HRS cells within a polymorphous inflammatory background). In this multi institutional study of 77 patients with CHL-RT (27 type 1 and 50 type 2), we characterized immune evasion markers, PD-L1/PD-L2 copy number alterations, tumor microenvironment, and performed targeted next-generation sequencing on 37 CLL/SLL samples. HRS cells in CHL-RT displayed immune evasion phenotypes similar to de novo CHL, though PD-L1 expression was lower in type 1 cases. PD-L1/PD-L2 gain/polysomy were frequent (83.3%). CLL/SLL with CHL-RT harbored increased mutations in XPO1, FBXW7, BIRC3, TRAF3, and HLA-A versus reference CLL/SLL. Similar mutational profiles, demographics, and survival outcomes support a biological continuum between type 1 and type 2 CHL-RT, with distinct genetic features in CLL/SLL predisposing to CHL transformation.

Background: Multiple myeloma (MM) remains incurable despite therapeutic advances, reflecting limited understanding of the molecular mechanisms underlying disease initiation and progression. MM develops through asymptomatic precursor stages, monoclonal gammopathy of undetermined significance (MGUS) and smouldering multiple myeloma (SMM). This study aimed to investigate protein changes associated with disease progression and, through a further integrative approach, to highlight molecular changes of potential predictive and/or therapeutic value. Methods: We performed a comparative proteomic analysis of 94 bone marrow-derived CD138+-selected plasma cell samples (29 MGUS, 20 SMM, and 45 MM) using LC-MS/MS. Differential protein abundance was assessed using pairwise Mann-Whitney U tests between groups, with Benjamini-Hochberg correction. Pathway enrichment, protein-protein interaction, and co-expression network analyses were also conducted. Selected proteins were further evaluated using public transcriptomic datasets and experimentally validated in independent samples by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Results: Following data processing, proteomic analysis identified 6,203 proteins. Pairwise comparisons revealed significant proteomic differences across disease stages, with 370 differentially abundant proteins exhibiting monotonic changes during disease progression. Pathway analysis showed that monotonically upregulated proteins were mainly associated with gene expression and cell proliferation, whereas downregulated proteins were linked to immune-related processes. Further co-expression network analysis, combined with criteria including detection frequency, biological relevance, and translational potential, highlighted a group of prioritised proteins. Representative examples include nucleolin (NCL) and U3 small nucleolar ribonucleoprotein IMP3 (IMP3), involved in nucleolar organisation, ribosome biogenesis and rRNA processing, as well as the immune-associated lactotransferrin (LTF) and serine protease cathepsin G (CTSG). Transcriptomic support and independent experimental validation by flow cytometry and ELISA confirmed the relevance of selected candidates. Conclusions: Taken together, our findings highlight coordinated changes in immune regulation, RNA processing and ribosome biogenesis during MM progression and identify candidate proteins and their networks, including the emerging pharmacologically tractable target NCL and the underexplored IMP3 of potential therapeutic relevance, opening new avenues for further investigation.

Waldenstrom macroglobulinemia (WM) is a rare indolent neoplasm characterized by presence of [&ge;] 10% lymphoid cells in BM that exhibit plasmacytoid or plasma cell differentiation that secretes an IgM monoclonal protein. This is a retrospective analysis of 89 patients of WM that describes the clinical and laboratory characteristics, treatment patterns and outcome of patients of WM. The median age of the entire cophort was 66 years with male predominance (67.4%). Most common presentations were symptoms pertaining to anemia (77.5%) and constitutional symptoms (33.7%). Median bone marrow lymphoplasmacytic cells were 41%. Positivity for MYD88 and CXCR4 mutations were seen in 81.8% and 2.4% cases. BR was the most common regimen used (52.8%). Overall response rates were seen at 87.8%. Median overall survival, progression free survival and time to next treatment is 8.49 years, 2.15 years and 3.88 years. BR regimen was associated with highest event free survival.

Diffuse Large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma in the Western world. First-line immunochemotherapy fails in approximately 30-40% of patients, with refractory and relapse patients presenting a dismal prognosis. Currently, these high-risk patients cannot be accurately identified at diagnosis. Using statistical modeling and machine learning approaches applied to large public DLBCL datasets, we identified a novel predictive signature based on the reactivation of eight normally silent tissue-dependent genes associated with survival. We then developed a multiplex RT-MLPseq based assay, compatible with formalin-fixed paraffin-embedded (FFPE) samples and transferable into routine clinical practice, enabling analysis of expression of these eight genes and validated their prognosis impact in an independent real-life cohort. This signature could be integrated with current prognostic indices and molecular classifications to improve patient stratification and guide treatment selection toward a personalized theragnostic approach, thereby enhancing management of non-responder patients. Data Sharing StatementFor access to original data, please contact: anouk.emadali@univ-grenoble-alpes.fr Key points- Ectopic activation of 8 tissue-specific genes defines a robust prognostic signature for survival stratification in DLBCL patients - A FFPE-compatible RT-MLPseq assay enables clinical use and improves risk stratification beyond IPI and COO, especially in high-risk patients

PurposeClinical translation of CAR T cell therapies has accelerated, yet preclinical evidence still often originates from single-center studies lacking sufficient robustness. Preclinical confirmatory multicenter studies have been proposed to improve the translational success, but their feasibility in cellular therapies remains unexplored. MethodsWe performed a confirmatory multicenter study validating C-C-motive-receptor-8 (CCR8) overexpression in CAR T cells--a strategy previously shown to enhance solid tumor infiltration. In vitro experiments covering activation, cytotoxicity, and migration using three CAR constructs were conducted across two centers with harmonized materials, preregistered protocols, randomization, and blinding. ResultsThe data from the two centers confirmed key findings of the exploratory study: CCR8 overexpression in anti-EpCAM and anti-mesothelin CAR T cells leads to enhanced selective migration towards a CCL1-gradient, while not compromising antigen-specific T cell activatory capacity and cytotoxicity in vitro. The study furthermore broadened the applicability of CCR8 overexpression to anti-CEA CAR T cells. ConclusionsThis first-of-its-kind preclinical confirmatory CAR T study demonstrates the feasibility of a multicenter confirmation in cellular therapy, with technical and logistical challenges resolved through transparent communication between all parties involved. Both exploratory and confirmatory studies aim to downselect CAR candidates with the highest clinical success potential, as they compete for limited resources in preclinical research. It is therefore mandatory to clarify the extent of replications required to validate the experimental methodology and identify CAR candidates with most likelihood of success. TRANSLATIONAL RELEVANCEPreclinical evidence for novel CAR T cell therapeutic strategies relies mostly on exploratory single-center studies lacking robustness, with recent findings substantiating their limited predictive value for cellular therapies tested outside hematology. Here, the function of CCR8-armored CARs in vitro was confirmed in a preclinical confirmatory multicenter study, demonstrating the feasibility of such studies in adding value to the transition of preclinical concepts to clinical development. Our first-of-its-kind study may contribute to define new routes for preclinical testing and further raises the general question of what level of preclinical evidence is reasonably achievable in an academic context. It indicates the need for strong collaborative efforts to realize dedicated preclinical infrastructure for clinical translation of reprogrammed immune cellular therapeutics.

Therapy-driven genomic changes in multiple myeloma (MM) remain poorly defined. We analyzed whole-genome sequencing (WGS) data from relapsed/refractory MM (rrMM, N=386) and identified regional 1p31.1-p12 (hereafter 1pCEN, a region proximal to the centromere) loss-of-heterozygosity (LOH) as the only enriched aberration showing strong therapy-associated clonal selection (clonal timing rank fold-change = 3.7, P<2.2x10-16). This event showed enriched co-occurrence with 1qGain (OR = 2.3 (1.5-3.8), P=2x10-4) forming a recurrent "double-hit" in rrMM. To validate the clonal selection process, we examined three longitudinal cohorts (180 patients, 390 samples) and confirmed clonal expansion of 1pCEN and consistent prevalence of the 1pCEN+1q double-hit (20-24%). Survival analyses demonstrated significantly reduced progression-free survival in rrMM patients with this double-hit compared with those without. Comparison with a large newly diagnosed MM (ndMM) cohort confirmed previously-described 1p32 LOH is the prognostic locus at baseline, whereas 1pCEN is therapy-selected and largely independent of the 1p32 locus. Thus, 1pCEN+1q represents a recurrent double-hit event that clonally emerges in rrMM, conferring selective advantage under drug exposure and is distinct from the ndMM high-risk markers defined by current consensus guidelines. These findings nominate 1pCEN as a new genomic biomarker in rrMM and 1pCEN+1q may help patient stratification for therapeutic monitoring. Key PointsA therapy-driven common genomic double-hit (1p31.1-p12 LOH with 1q gain) clonally emerges in relapsed/refractory myeloma.

Myeloproliferative neoplasms (MPNs) are characterized by progressive myelofibrosis that drives morbidity and mortality. Liquid biopsy approaches to noninvasively monitor fibrotic progression remain limited. We performed comparative transcriptomic profiling of CD45-depleted platelet-enriched and CD45+ leukocyte-enriched fractions from matched peripheral blood samples of 76 individuals (27 primary myelofibrosis, 17 polycythemia vera, 14 essential thrombocythemia, 18 healthy controls). Platelet RNA sequencing was performed in 2018-2020 on Illumina HiSeq 4000, while WBC RNA sequencing was conducted in 2023 on Illumina NovaSeq 6000 from cryopreserved CD45+ enriched fractions of specimens obtained at the identical time and from the same blood sample as the platelet RNA. Despite comparable library preparation protocols and higher sequencing depth in WBC samples, platelet transcriptomes exhibited 5.1-fold more differential expression in myelofibrosis (3,453 versus 681 genes, adjusted p<0.05, |log2FC|>1). Platelet signatures were enriched for proteostasis pathways including endoplasmic reticulum stress and unfolded protein response, reflecting megakaryocyte dysfunction in the fibrotic bone marrow niche. WBC signatures predominantly featured immune activation and proliferative pathways, indicating systemic inflammatory responses. Multinomial LASSO classification demonstrated superior performance of platelet-based models for myelofibrosis diagnosis (AUROC 0.85) compared to WBC-based (AUROC 0.77) or clinical models (AUROC 0.59). Combined platelet+WBC models did not improve performance (AUROC 0.80), indicating complementary but non-additive information. These findings establish platelet transcriptomic profiling as a superior noninvasive biomarker platform for monitoring myelofibrosis in MPNs, capturing megakaryocyte-driven fibrogenesis with greater sensitivity than peripheral leukocyte-based approaches. HighlightsUsing matched WBC and platelet RNA-seq from MPN patients, we identify myelofibrosis-associated transcriptomic signatures specifically enriched in platelets. Multinomial LASSO modeling highlights platelet-derived gene expression as a dominant and predictive biomarker of myelofibrosis, outperforming clinical parameters and WBC signatures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/714941v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1d695aborg.highwire.dtl.DTLVardef@fc250forg.highwire.dtl.DTLVardef@1e52e8eorg.highwire.dtl.DTLVardef@15378e3_HPS_FORMAT_FIGEXP M_FIG C_FIG

Key PointsO_ST_ABSQuestionC_ST_ABSDoes the circadian timing of stem cell infusion influence the risk of aGVHD after allo-PBSCT? FindingsIn this randomized prospective clinical trial that included 198 patients, infusion stem cell at 12:00 pm at noon was associated with a significantly lower incidence and less severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. MeaningScheduling stem cell infusion at an earlier time-of-day may reduce aGVHD risk after allo-PBSCT. IMPORTANCEAcute graft-versus-host disease (aGVHD) remains a major complication following allogeneic peripheral blood stem cell transplantation (allo-PBSCT), compromising patient survival and quality of life. OBJECTIVETo evaluate the effect of stem cell infusion time-of-day on aGVHD after allo-PBSCT. DESIGNA multicenter, randomized, open-label, phase 3 clinical trial was conducted from March 18, 2024, through June 11, 2025, with follow-up through December 31, 2025 (median, 462 days among survivors). SETTINGSix transplantation centers in China. PARTICIPANTSPatients aged 12 to 60 years with malignant hematologic diseases undergoing first allo-PBSCT were screened; 198 eligible patients were randomized. INTERVENTIONSPatients were randomly assigned in a 1:1 ratio to receive stem cell infusion at either 12:00 pm at noon ({+/-} 0.5 hour) or 6:00 pm ({+/-} 0.5 hour). MAIN OUTCOMES AND MEASURESThe primary end point was the cumulative incidence of grade II-IV aGVHD within 100 days after transplantation. Secondary end points included grade III-IV aGVHD, hematopoietic recovery, transplant-related mortality (TRM), relapse, and survival outcomes. RESULTSAmong 198 randomized patients (median age, 38 years; 119 [60.1%] male), grade II-IV aGVHD within 100 days occurred in 11 of 99 patients (11.1%) in the 12:00 pm group and 22 of 99 patients (23.2%) in the 6:00 pm group. The cumulative incidences of grade II-IV and III-IV aGVHD were significantly lower in the 12:00 pm group (II-IV: 11.1% [95% CI, 5.9%-18.2%] vs 23.2% [95% CI, 15.4%-32.0%], P = 0.029, hazard ratio, 2.18 [95% CI, 1.06-4.48]; III-IV: 2.0% [95% CI, 0.4%-6.5%] vs 12.2% [95% CI, 6.7%-19.5%], P = 0.006, hazard ratio, 6.25 [95% CI, 1.39-28.15]). There were no significant differences in hematopoietic recovery, TRM, or relapse between groups. The estimated probability of GVHD-free, relapse-free survival (GRFS) at 360 days favored the 12:00 pm group (66.7% [95% CI, 56.2%-75.2%] vs 56.5% [95% CI, 46.1%-65.5%]). CONCLUSIONS AND RELEVANCEStem cell infusion at 12:00 pm was associated with a lower incidence and severity of aGVHD compared with infusion at 6:00 pm, without influencing engraftment or relapse. Optimization of infusion timing may represent a simple strategy to reduce aGVHD risk. TRIAL REGISTRATIONClinicalTrials.gov Identifier: NCT06294678.

Background: Large language models (LLMs) perform well on standardized medical exam questions, but their reliability for complex hematology decision making is uncertain. We compared four general-purpose LLMs (GPT-4o, GPT-o3, Claude Sonnet 4, and DeepSeek-V3) with a Virtual MDS Panel (VMP), a coordinated multi-agent AI system in which domain-specialized, rule-bound software agents (WHO/ICC guidelines; IPSS-R/IPSS-M; NCCN) collaborate to generate tumor-board-level recommendations. Methods: Each model generated diagnostic, prognostic, and treatment recommendations for 30 myelodysplastic syndrome cases. Nine international MDS experts from five institutions, blinded to model identity, completed 3,000 structured ratings using 5-point Likert scales for diagnosis, prognosis, and therapy and classified errors by severity. Results: General-purpose LLMs achieved modest expert ratings (overall mean scores: 3.7 for GPT-o3, 3.2 for GPT-4o, 3.1 for DeepSeek, and 3.0 for Claude) and contained major factual errors in at least 24% of responses. The VMP increased the proportion of outputs rated 4 or higher to 87% (vs. 34-66% for general-purpose models), improved mean scores to 4.3 overall (4.3 for diagnosis, 4.4 for prognosis, and 4.1 for therapy), and reduced major errors to 8%. Conclusions: In this blinded evaluation of 30 complex MDS cases, general-purpose LLMs produced clinically important errors at rates that raise safety concerns for autonomous hematology decision making. The VMP, a rule-bound, multi-agent architecture, approached expert-level accuracy supporting its potential role as an effective decision-support tool for MDS in the future.

BackgroundChronic myelomonocytic leukemia (CMML) is a clinically heterogeneous myeloid malignancy with limited therapeutic options and suboptimal risk stratification. Although single-cell RNA sequencing has refined disease classification through gene expression profiling, post-transcriptional mechanisms--particularly adenosine-to-inosine (A-to-I) RNA editing--remain unexplored at single-cell resolution. We hypothesized that cell-specific RNA editing programs contribute to CMML heterogeneity and define distinct, clinically actionable cellular states in CMML. MethodsWe developed a single-cell-aware computational framework for high-confidence identification and quantification of RNA editing events. Candidate sites were detected at pseudo-bulk depth using stringent filters and subsequently quantified at single-cell resolution. The pipeline incorporated dual alignment, barcode correction, artifact removal, and exclusion of genomic variants to ensure specificity. We applied this framework to discovery and independent validation CMML cohorts. Editing-defined cellular states were identified by unsupervised clustering of single-cell editing profiles and evaluated for associations with clinical stage, TET2 status, survival, and response to hypomethylating agent (HMA) therapy. Regulatory mechanisms were assessed by analyzing ADAR1/ADAR2 expression and relationships between editing levels and target gene expression. ResultsWe identified 3,326 high-confidence A-to-I RNA editing sites and delineated reproducible editing-defined cellular states. A granulocyte-monocyte progenitor-like editing state (edClu1_sub0) aligned with an inflammatory, monocytic-biased transcriptional program and was significantly associated with adverse survival, advanced-stage disease and TET2-mutant CMML, supporting it as a high-risk biomarker-defined subpopulation. In contrast, states such as edClu3 and edClu6 were enriched in earlier-stage, TET2-wild-type CMML and correlated with improved outcomes. Editing-defined states demonstrated systematic remodeling following HMA therapy, indicating treatment-responsive post-transcriptional programs. The high-risk state exhibited elevated ADAR1 and reduced ADAR2 expression, suggesting enzyme-specific regulatory imbalance as a potential therapeutic vulnerability. Integrative analyses further nominated immune-related genes--including LAPTM5, CTSS, and CD83--as CMML-specific oncogenic RNA editing targets, with coordinated increases in editing and expression within the aggressive state. ConclusionsRNA editing represents a clinically informative and mechanistically relevant layer that refines CMML stratification at single-cell resolution, independent of gene expression. These findings provide a framework for integrating post-transcriptional regulation into precision oncology and highlight RNA editing signatures as biomarkers for risk assessment, treatment monitoring, and therapeutic targeting in hematologic malignancies.

TP53 mutations represent one of the strongest adverse prognostic factors in myelodysplastic neoplasms (MDS). While multi-hit TP53 (TP53multiHit) alterations uniformly lead to very poor outcomes, the prognostic relevance of monoallelic TP53 (TP53mono) mutations remains controversial. TP53 variants can cause loss-of-function, dominant-negative, or gain-of-function effects. We hypothesized that functional heterogeneity among TP53 variants contributes to the variable clinical behavior observed in monoallelic TP53-mutated MDS. Therefore, we analyzed pretreatment samples from 4,505 patients with MDS from two independent cohorts (IWG, n=3,173; J-MDS, n=1,332), including 271 patients with TP53mono and 499 with TP53multiHit. Functional annotation of TP53 variants was performed using a previously published phenotype score (PS) derived from saturation mutagenesis screens, capturing dominant-negative and loss-of-function effects. Median overall survival (OS) differed significantly by TP53 allelic state (TP53 wild-type (TP53wt) 42.4 months; TP53mono 22.9 months; TP53multiHit 9.2 months; p < 0.001). Within the TP53mono subgroup, functional annotation identified marked heterogeneity. Patients with high PS ([&ge;]7) showed significantly inferior OS compared with those with low PS (median OS: 13.8 vs. 39.2 months; HR 1.68, 95% CI 1.16-2.42; p = 0.006), particularly for IPSS-R and IPSS-M low-risk cases. Combining PS and variant allele frequency (VAF) further improved risk stratification. TP53mono patients with PS [&ge;]7 and VAF [&ge;]22% had outcomes comparable to TP53multiHit (median OS: 8.8, p = 0.2), whereas those with PS <7 and VAF <22% exhibited survival similar to TP53wt (median OS: 49.7, p = 0.9). Overall, functional annotation of TP53 variants refines prognostication in TP53mono-mutated MDS and may enhance individualized risk assessment.

IntroductionImmune checkpoint inhibitors (ICIs) enhance antitumor responses by blocking inhibitory receptors, including PD-1 and CTLA-4. Overactivation can trigger systemic toxicity akin to autoimmune diseases, including kidney manifestations. We sought to 1) profile immune signaling and 2) interrogate potential mechanisms of ICI-related kidney injury in a Human Immune System (HIS) tumor-bearing mouse model treated with nivolumab and ipilimumab. MethodsImmunodeficient BRGS (BALB/c-Rag2nullIl2r{gamma}nullSirpNOD) neonates were engrafted with human CD34+ cells to generate HIS-BRGS mice. Human MDA-MB-231 tumor cells were implanted subcutaneously; once tumors reached [~]150 mm3, mice received weekly intraperitoneal vehicle (PBS) or ICI (nivolumab 20 mg/kg + ipilimumab 10 mg/kg) for 4 weeks (Veh BRGS n=4; ICI BRGS n=6; Veh HIS-BRGS n=7; ICI HIS-BRGS n=7). Kidneys were evaluated by histopathology (H&E, TEM), flow cytometry for human immune phenotypes, multiplex ELISA (80 human proteins; 10 injury biomarkers), bulk RNA sequencing, and targeted qPCR. Pearson correlations identified predictors of histopathological injury. ResultsRenal vasculitis and interstitial nephritis were observed only in ICI-treated HIS-BRGS mice. These kidneys showed a shift toward CD4+ T-cell enrichment with an increased TNF- production capacity compared to CD8+ counterparts. Toxicity was accompanied by increased renal concentrations of human cytokines, chemokines, and soluble receptors. ICI treatment significantly elevated serine proteases (Granzyme A/B) and NGF-{beta}, while decreasing IL-4. Interstitial nephritis correlated with renal PD-1 and MIF. Renal vasculitis correlated with kidney PD-1, CCL1, MIF, Granzyme A, IL-15, and BAFF. Traditional injury biomarkers (KIM-1, NGAL) remained unchanged; however, a trending decrease in EGF was observed. ConclusionsOur study suggests that shifts in human T-cell populations and specific immune proteins could serve as promising biomarkers and mechanistic targets for ICI nephrotoxicity. The tumor-bearing HIS-BRGS mouse model reproducibly recapitulates the histopathological and immunological features of human ICI-induced nephrotoxicity and represents a validated preclinical platform for testing novel therapeutic interventions to preserve kidney function during cancer immunotherapy. Translational StatementImmune checkpoint inhibitor (ICI)-associated nephrotoxicity occurs in up to 25% of treated patients, yet the immunological mechanisms driving renal injury remain poorly characterized due to the scarcity of human biopsy material and the absence of robust preclinical models that recapitulate human immune responses. This study demonstrates that tumor-bearing humanized immune system (HIS) mice treated with combined nivolumab and ipilimumab reproducibly develop renal vasculitis and interstitial nephritis mediated by a human CD4+ T cell-dominant infiltrate, mirroring the clinicopathological features reported in patients with ICI-associated acute kidney injury. By integrating histopathology, flow cytometry, multiplex proteomics, and transcriptomics, we identify a coordinated immune network, including IL-15, CCL1, MIF, GZMA, and BAFF, that correlates with the severity of renal pathology and represents tractable mechanistic targets and candidate biomarkers. These findings provide a validated preclinical platform for dissecting irAE mechanisms and testing novel therapeutic strategies to preserve kidney function during cancer immunotherapy.

Chimeric Antigen Receptor (CAR) T cells are now established as therapies for some haematological malignancies. While lentiviral or {gamma}-retroviral vectors are commonly used for CAR delivery due to their efficiency and stable integration, supply constraints have created bottlenecks to wider applications and access. Alternatively, genome editing tools such as CRISPR-Cas9 can insert CAR genes by homology-directed repair (HDR) into specific genomic loci. Universal donor CAR-T cells devoid of endogenous TCR{beta} after CRISPR-Cas9-mediated editing of the T cell receptor alpha (TRAC) locus are being investigated for more cost-effective, off-the-shelf therapies. Targeting insertion of CARs into the TRAC locus places transcription under the control of native regulatory machinery while simultaneously disrupting endogenous TCR{beta}, and this has been reported to reduce exhaustion and extend persistence in modelling studies using humanised mice. We compared anti-CD20 CAR-T cells, generated with CAR inserts at either TRAC or CD3{zeta} loci using entirely virus-free manufacture, and universal CAR20-T cells generated using existing lentiviral procedures and CRISPR/Cas9 knockout. While non-viral cell yields were lower than lentiviral products cytotoxic function in vitro was comparable between groups. Studies in humanised murine models of leukaemia inhibition found non-viral CAR20-T cells were generally less efficacious than LV-CAR20 and exhibited more exhausted phenotypes. Non-viral approaches offer the prospect of sophisticated editing and precise CAR insertion but careful preclinical evaluation and well-designed clinical trials benchmarked against lentiviral approaches are recommended.