The theoretical possibility for leukemia stem cells (LSCs) to produce both leukemia blasts and dysfunctional immune cells remains underexplored. Here, we investigate three major fusion transcription factor (fTF)-driven acute myeloid leukemia (AML) subtypes [RUNX1(CBFα)::RUNX1T1, PML::RARA, and CBFB::MYH11] using two optimized single-cell RNA-sequencing technologies to trace fTF expression in 24 de novo AML patients. We demonstrate that the fTFs are widely expressed not only in leukemia blasts but also in differentiated myeloid and lymphoid cells, indicating hematopoietic stem cells or multipotent progenitors (HSCs/MPPs) as LSCs that propagate altered cellular differentiation hierarchies, including immune cells. DNA-FISH confirms the presence of fTFs in T lymphoid and erythroid cells, and targeted sequencing of secondary mutations in sublineages of cells corroborates hierarchical and stepwise leukemogenesis. By tracking RUNX1::RUNX1T1-expressing cells in patients with or without relapse post-frontline chemotherapy, we highlight the necessity of eradicating LSCs to achieve sustained long-term complete remission and restore a functional immune system capable of suppressing residual disease over time. Comparative single-cell transcriptome analyses further reveal that fTFs are associated with AML subtype-specific differentiation defects in both innate and adaptive immune compartments, suggesting an altered landscape of immune cell-cell communication networks that may facilitate the survival and proliferation of leukemic blasts. Through the examination of intercellular communications among various putative fTF+ and normal cell populations, we developed a ligand-receptor (L-R)-based risk-scoring model with independent prognostic value. Collectively, these findings provide insights into the cells of origin of LSCs and the implications of fTF expression for the immune landscape of AML.

Core binding factor acute myeloid leukemia (CBF-AML) is defined by t(8;21) or inv. (16), which give rise to the RUNX1::RUNX1T1 and CBFβ::MYH11 fusion genes, respectively. CBF-AML is a favorable-risk AML subtype, yet differences in mutation profiles, measurable residual disease (MRD) response, and relapse risk may warrant tailored treatment approaches. We reviewed adult AML patients treated at five transplant centers across China and identified 825 de novo CBF-AML cases, of which 779 met our eligibility criteria: 536 harbored the RUNX1::RUNX1T1 fusion and 243 the CBFβ::MYH11 fusion. The 3-year overall survival (OS) was 80.6% for RUNX1::RUNX1T1 and 90.2% for CBFβ::MYH11 cases (p = 0.011). Among patients with RUNX1::RUNX1T1, those achieving MRD negativity after two consolidation cycles (PC2) had significantly better OS than nonresponders (86.3% vs. 76.5%, p = 0.008); no difference was observed for CBFβ::MYH11 patients. For RUNX1::RUNX1T1 nonresponders, allogeneic hematopoietic cell transplantation (allo-HCT) in first complete remission (CR1) reduced relapse (CR1-HCT vs. non-CR1-HCT, 8.3% vs. 18.9%; p = 0.024) and improved OS (CR1-HCT vs. non-CR1-HCT vs. chemotherapy, 85.8% vs. 71.4% vs. 64.9%; p < 0.001). In CBFβ::MYH11 patients, deferring allo-HCT until second complete remission (CR2) was associated with comparable outcomes. In RUNX1::RUNX1T1 patients, older age, elevated initial white blood cell count, lower hemoglobin, lower initial fusion transcript load, and the presence of FLT3-ITD or KIT D816/D822 mutations were associated with an increased likelihood of PC2 nonresponse. Based on these variables, we developed a weighted scoring system with good discrimination to identify RUNX1::RUNX1T1 patients at high risk of PC2 nonresponse.

Acute myeloid leukemia/myelodysplastic syndromes (AML/MDSs) carrying p53 mutations are refractory to various standard therapies. Arsenic trioxide (ATO) may be effective in restoring function to p53 structural mutants. Here, we report that mutant p53 rescued by ATO treatment strengthened interferon responses triggered by the DNA hypomethylating agent decitabine by transactivating interferon regulatory factor 7 (IRF7) directly. Decitabine also increased the transactivation activity of ATO-rescued mutant p53 by inducing p53-serine-20 phosphorylation and blocking p53-inhibitory mouse double minute 2 homolog (MDM2). ATO and decitabine together killed p53-mutant AML cells and suppressed tumor growth in cell line-derived xenografts. In a first-in-human pilot clinical trial for testing the combination of ATO and decitabine (PANDA-T0 trial, NCT03855371), which enrolled five patients with AML/MDS harboring p53 structural mutations, the ATO and decitabine regimen produced manageable adverse events, and four of the five treated patients achieved complete remission at the level of the bone marrow, associated with p53 activation and interferon response. In 103 p53-mutant patients whose samples were deposited in Ruijin AML/MDS sample repository, 48 distinct p53 missense mutants were identified, 21 of which were classified as ATO and decitabine regimen applicable because of their competencies in activating p53 and interferon responses upon cotreatment. This study establishes an alternative treatment regimen for patients with p53-mutant AML/MDS and provides a proof-of-concept framework for p53-targeted therapy that differentiates between p53 mutations.

The ZMYND11::MBTD1 fusion, resulting from t(10;17)(p15;q21), is a rare recurrent genetic alteration in acute myeloid leukemia (AML). Its clinical and prognostic features in adolescent and adult patients remain poorly characterized. This study aimed to delineate the clinicopathological, immunophenotypic, and molecular features, as well as treatment responses and survival outcomes, of patients harboring this fusion. We retrospectively analyzed five newly diagnosed AML patients aged ≥ 16 years with ZMYND11::MBTD1 fusion treated at our institution between 2020 and 2024. The five molecularly confirmed institutional cases were analyzed together with nine previously reported adult cases with t(10;17)(p15;q21)/ZMYND11::MBTD1-associated AML, yielding an integrated cohort of 14 patients. All institutional cases were identified via targeted RNA sequencing with PCR confirmation. Immunophenotyping, cytogenetics, treatment response, and overall survival (OS) were evaluated. All five institutional cases exhibited a consistent RAM-like immunophenotype characterized by bright aberrant CD7 and CD56 expression, positivity for CD33 and CD117, dim CD45, and absent or reduced HLA-DR and CD38. Conventional karyotyping detected t(10;17) in only one of five cases. In the institutional cohort, the complete remission (CR) rate after one induction cycle was 40

ABSTRACT:GPRC5D has emerged as a promising therapeutic target in relapsed/refractory multiple myeloma (R/R MM), particularly following progression after B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell (CAR-T) therapies. RD118 is a novel CAR-T therapy incorporating a fully human single-domain antibody fragment targeting GPRC5D. In this phase 1 study, 18 R/R patients (17 with MM and 1 with a history of primary plasma cell leukemia) received a single infusion of RD118 at 1.0 × 106, 2.0 × 106, or 3.0 × 106 CAR+ T cells per kg. At a median follow-up of 17.0 months, the overall response rate (ORR) was 94.4%, including 72.2% complete or stringent complete responses. Among the 7 patients previously exposed to BCMA-directed CAR-T therapy, ORR reached 85.7%. Median progression-free survival (PFS) was 18.2 months (95% confidence interval, 14.4 to not estimable), with 12-month PFS and overall survival rates of 82.1% and 93.3%, respectively. Cytokine release syndrome occurred in 88.9% of the patients, primarily grade 1 to 2. One patient developed grade 3 immune effector cell-associated neurotoxicity, which resolved within 72 hours. No cerebellar toxicities or treatment-related deaths were reported. These findings support that RD118 is a highly effective and safe therapeutic option for heavily pretreated R/R MM. This trial was registered at www.clinicaltrials.gov as #NCT05759793 and #NCT05219721.

Cancer differentiation therapy aims to induce the maturation of neoplastic cells, but the mechanisms regulating cell fate decisions in oncogenic contexts remain unclear. In this study, we integrated single-cell chromatin accessibility and single-cell transcriptome analyses to explore the regulatory trajectories of a classical PML/RARα+ acute promyeloid leukemia (APL) cell line (NB4) post treatment by all-trans-retinoid acid (ATRA). Our findings indicated that ATRA activated specific PML/RARα-target enhancers to trigger a regulatory circuit composed of a positive feedforward gene regulatory circuit involving two transcription factors, SPI1 and CEBPE. This regulatory circuit was both necessary and sufficient to drive NB4 cells through an intermediate cell fate decision point to initiate terminal granulopoiesis. Moreover, ectopic expression of SPI1 and CEBPE promoted granulocytic differentiation in non-APL leukemia cell lines HL60 and K562. Our study sheds mechanistic insights into the differentiation trajectories induced by ATRA and illustrates a gene regulatory circuit that could be widely applied to promote differentiation of leukemia cells.

Abstract Background For newly diagnosed acute myeloid leukemia (ND-AML), the traditional “3+7” induction is still the foundation of intensive chemotherapy. For certain subgroups, the combination of targeted drug can improve response rate and long-term survival. As previous reported in RJ-AML 2014 and 2016 trial [Am J Hematol. 2022;97(1):43-51; 65th ASH Annual Meeting and Exposition, San Diego, CA], day 5 peripheral blast clearance rate (D5-PBCR) can be used as an indicator of early treatment response. For patients in the D5-PBCR (+) group, addition of homoharringtonine can benefit favorable and intermediate risk groups. While for adverse-risk and FLT3-ITD mutated cases, other interventions were needed. Patients and Methods IA+X 2024 is a multicenter, non-randomized, phase II clinical trial, conducted to evaluate the efficacy of targeted drug (drug “X”) in combination with “3+7” regimen for ND-AML (ClinicalTrials.gov NCT06652685). Patients receive IA-10 regimen (idarubicin 10mg/m2 on days 1–3 and cytarabine 100mg/m2 on days 1–7) as the initial induction. On day five of induction, D5-PBCR will be tested. Patients will be assigned to different arms according to the results of molecular tests and D5-PBCR. For CBF-AML patients, no interventions are needed. For FLT3-ITD mutated patients, a combination of gilteritinib is recommended (induction and consolidation: 80mg/d on days 8-21). All other D5-PRCR (+) patients will receive Venetoclax (VEN) addition (induction: 100mg on day 6, 200mg on day 7, and 400mg on days 8–13; consolidation: 400mg/d on days 8-14). The primary end point is composite complete remission (CRc) rate. We hypothesized that the CRc rate may increase by 15% (from 67% to 82%) with the addition of drug “X”. As designed using the single-arm, Simon two-stage model, twenty-nine patients were accrued in the first stage of analysis and if more than 20 CRc were observed, 31 additional patients will be included. The secondary end points include total CRc rate, overall survival (OS) and event-free survival (EFS). Consolidation therapy will be assigned according to the patients' ELN 2022 risk stratification. Here, we report the interim analysis. Results Between August 2024 and April 2025, a total of 115 patients underwent screening, 97 were included in the intention-to-treat (ITT) population and received IA-10 induction from 5 sites in China. Twenty-seven patients were classified as CBF-AML (14 cases with RUNX1::RUNX1T1 fusion, 13 with CBFβ::MYH11 fusion). The analysis of CBF-AML subgroup showed 100% CR rate after one cycle of induction with 50% and 100% MRD negativity by flow cytometry (<0.01%) in RUNX1::RUNX1T1 and CBFβ::MYH11 fusion groups, respectively. D5-PBCR were analyzed in 70 patients and 42 (60%) had D5-PBCR (+). Baseline characteristics, including age, sex, WBC, bone marrow blast counts, and cytogenetic parameters were similar between two groups. Patients with NPM1 mutation were more sensitive to IA-10, while TP53 mutation was more common in D5-PBCR (+) group. Of the 42 D5-PBCR (+) patients, 3 cases discontinued IA and 6 were unfit for drug “X” additional. Of the 28 D5-PBCR (-) patients, 2 patients discontinued IA and 2 were unfit for gilteritinib combination. The overall composite CR rate after one course of induction was 76.2% in the ITT cohort, and 87.7% in the Per-Protocol (PP) cohort. In PP population, CR rates were 95.8% and 81.8% for D5-PBCR (-) and D5-PBCR (+) groups, respectively. No early death events occurred within 60 days of induction. All patients in the 2022 ELN favorable-risk group obtained CR. FLT3-ITD mutated patients (15 in ITT and 12 in PP) showed high response with IA+gilteritinib, which is also observed in patients with NUP98 fusion. However, there is a certain degree of prolongation in the duration of bone marrow suppression. While subgroup analysis showed that only one patient with TP53 mutation achieved CR after one cycle of IA+VEN induction. Even after receiving the second course of induction, only 2 patients achieved remission, with the remaining 5 patients in non-remission. Conclusion The IA+X 2024 protocol has currently met the requirements for the interim analysis as designed, and enrollment is planned to continue. All patients in the favorable-risk group achieved CR after one course of induction. And for patients with TP53 mutation, a new re-induction regimen needs to be designed.

KMT2A-altered acute myeloid leukemia (AML) comprises rearrangements (KMT2A-r), partial tandem duplications (KMT2A-PTD), and dual alterations (KMT2A-r/PTD). In this study of 125 patients, these subgroups exhibited distinct molecular profiles: KMT2A-r cases were enriched in RAS pathway mutations, whereas KMT2A-PTD showed a higher burden of epigenetic alterations. Although overall survival (OS) and event-free survival (EFS) did not differ significantly between subgroups, prognosis was strongly influenced by fusion partners. MLLT3/ELL-rearranged cases showed superior outcomes, but concurrent KMT2A-PTD abrogated this survival advantage, AFDN and other fusions showed poor outcomes. We therefore propose a revised three-tier risk model integrating fusion partner and PTD status, which significantly stratified patient outcomes. The intermediate-risk group (MLLT3/ELL without PTD) had a 3-year OS of 78.1

Abstract Introduction: Core binding factor acute myeloid leukemia (CBF-AML), defined by t(8;21)/RUNX1::RUNX1T1 or inv (16)/CBFβ::-MYH11, is categorized as favorable-risk due to its high remission rate and better relapse-free survival. We aimed to delineate the similarities and differences in treatment response and prognosis between AML patients with RUNX1::RUNX1T1 and CBFβ::MYH11 fusions. Methods: 779 de novo CBF-AML patients (511 RUNX1::RUNX1T1, 225 CBFβ::MYH11) were enrolled. Measurable residual disease (MRD) was assessed via multi-parameter flow cytometry (MFC-MRD) and quantitative polymerase chain reaction (Mol-MRD) during/after treatment. Responders are defined as achieving MRD negativity (MFC-MRD 0.01% aberrant cells and Mol-MRD > 3-log reduction) after the second consolidation cycle (C2). Otherwise, it is considered positive. Survival analyses employed Kaplan-Meier and landmark methods (3-month cutoff [corresponding to the median time of C2 completion] for immortal time bias). Treatment response comparisons used time-dependent Cox models, while relapse risks were evaluated via the Prentice, Williams and Peterson (total times) (PWP-TT) modeling. Logistics regression models were used to develop the predictive model for the responses after C2. Results: With a median follow-up of 34.6 months (range: 3.9-106.2), early mortality by C2 was lower in RUNX1::RUNX1T1 (4.7%, n=25) versus CBFβ::MYH11 (7.1%, n=18, P = 0.042) patients. The 3-year overall survival (OS) was significantly better in patients with CBFβ::MYH11 compared to RUNX1::RUNX1T1 (90.2% vs 80.6%, P = 0.011). 695 patients were evaluable for C2 assessment, with 42.5% with RUNX1::RUNX1T1(n = 206) and 33.2% with CBFβ::MYH11 fusions (n = 74) as responders. Landmark analysis showed superior 3-year OS for RUNX1::RUNX1T1responders vs. non-responders (86.3% vs. 76.5%, P = 0.008), however, responders and non-responders have comparable survival outcomes in patients with CBFβ::MYH11 (95.1% vs. 88.1%, P= 0.49). Multivariable Cox model further identified Mol-MRD positivity (HR [95%CI]: 1.96[1.08–3.54], P = 0.026) and/or MFC-MRD positivity (2.79[1.61–4.85], P<0.001) at C2 as significant risk factors for RUNX1::RUNX1T1 survival, with no associations in CBFβ::MYH11. PWP-TT model showed that non-responders at C2 were related to more relapses in the RUNX1::RUNX1T1 patients (Mol-MRD positivity: 2.126[1.181-3.828], P = 0.012; MFC-MRD positivity: 2.855[1.733-4.704], P < 0.001), but not in patients with CBFβ::MYH11 fusion. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was a protective factor for long-term survival (HR [95%CI]: 0.391 [0.206-0.740], P = 0.004) with the multivariable Cox model and relapse (HR [95%CI]: 0.565 [0.386-0.828], P = 0.003) with the PWP-TT model in RUNX1::RUNX1T1 patients, but not for CBFβ::MYH11 patients. Integration of landmark analysis and time-dependent cohort allocation, we found that for RUNX1::RUNX1T1 non-responders, allo-HCT provided a superior 3-year OS compared with chemotherapy (81.9% vs. 56.4%, P < 0.001), which was different from non-responders with CBFβ::MYH11. Responders with RUNX1::RUNX1T1 or CBFβ::MYH11 had comparable outcomes when consolidated with allo-HCT or chemotherapy. Transplant in first CR provided better outcomes for RUNX1::RUNX1T1 (3-year OS: CR1-HCT 85.8% vs. non-CR1-HCT 71.4%, P < 0.001; relapses: CR1-HCT 8.3% vs. non-CR1 18.9%, P = 0.024). Consolidation therapies did not show significant impacts on long-term outcomes in CBFβ::MYH11 patients (3-year OS: Chemotherapy vs. non-CR1-HCTvs. CR1-HCT: 89.9% vs. 90.7% vs. 92,2%, P = 0.871). At last, we developed a comprehensive predictive model for C2 response incorporating clinical factors: (age < 40y [1 score], initial white blood count [<50 × 109/L: 1 score; 50-100: 2 score; > 100: 3 scores]; hemoglobin < 100 g/L [1 score]), mutational profiling (KIT D816/D822 variants [3 scores], other KIT mutations [1 score], FLT3-ITD [1 score]), as well as initial RUNX1::RUNX1T1/ABL value (<500%[4 scores], 500-1000%[3 scores], 1000-1500%[2 scores], ≥1500%[1 score]). A score ≥8 predicted non-response with 78.3% specificity and 57.4% sensitivity, effectively identifying high-riskRUNX1::RUNX1T1 AML cases needing urgent transplant evaluation. Conclusion: For CBFβ::MYH11 AML, C2 assessment offers minimal clinical significance, with allo-HCT reserved for CR2. In contrast, RUNX1::RUNX1T1 patients with post-C2 MRD positivity should receive allo-HCT at CR1.

Background Combining IKZF1plus genotyping and minimal residual disease status at three months of treatment helps refining risk stratification for adult BCR::ABL1–positive acute lymphoblastic leukemia (BCR::ABL1+ ALL) in our previous study. (Wang C, et al. Blood Cancer J. 2024 Apr 24;14(1):71). In parallel, ABL1 kinase domain mutations persist as a major therapeutic challenge. In spite of prognostic significance of the two factors individually, studies on the association of IKZF1plus genotype and ABL1 mutations remain limited. Aim To establish the prognostic profile and investigate underlying biological features of concomitant IKZF1plus genotype and ABL1 mutations in adult BCR::ABL1+ ALL. Methods Fron June 2014 to December 2024, newly diagnosed adult BCR::ABL1+ ALL patients (aged ≥18) treated in our institute were included. The patients received a TKI-based [imatinib or flumatinib (a second-generation TKI)] standardized VIP regimen (Vincristine/Idarubicin/Prednisone), and eligible patients were recommended to undergo allo-HSCT. At diagnosis, fresh bone marrow or blood samples were collected from patients with written informed consent. Multiplex ligation-dependent probe amplification (MLPA) was used for IKZF1plus genotyping at diagnosis, polymerase chain reaction was employed to routinely monitor MRD and ABL1 mutation status, and bulk RNA sequencing was utilized to characterize biological features. (Clinical Trial Registration Number: ChiCTRONRC-14004968, ChiCTR2100042248 and ChiCRT2100044308). Results According to MLPA results, 246 newly diagnosed BCR::ABL1+ ALL patients were classified into IKZF1plus (n = 98), and non-IKZF1plus genotype (n = 148), respectively. And based on ABL1 mutation analysis, the patients were categorized into three groups: T315I mutation (n = 62), mutations other than T315I (non-T315I, n = 34), and no mutation (n = 150), respectively. Compared to no mutation group, T315I mutation group exhibited a notably higher proportion of IKZF1plus genotype (P = 0.02), adults aged older than 65 years (P = 0.01), higher white blood cell counts (P = 0.02), MRD positivity at 3 months of treatment (P < 0.001), relapse (P < 0.001) and death (P < 0.001). Similarly, relative to non-T315I mutation group, T315I mutation group also demonstrated a significantly greater frequency of IKZF1plus genotype (P = 0.01) and relapse (P = 0.004). Till July 31, 2025, the overall median follow-up period was 31.8 months (range, 6.4-122.4). The 3-year overall survival (OS) of T315I mutation group was the worst [34.4% (95% CI: 19.8%-49.1%)], lower than non-T315I mutations group [61.7% (95% CI: 41.8%-81.7%), P = 0.07] or no mutation group [91.2% (95% CI: 85.8%-96.5%), P < 0.001]. Then focusing on IKZF1plus genotype, patients were stratified into three subgroups based on ABL1 mutation analysis:IKZF1plus/T315I (n = 34), IKZF1plus/non-T315I (n = 9), and IKZF1plus/no mutation (n = 55). IKZF1plus/T315I subgroup exhibited the lowest 3-year OS rate [21.2% (95% CI: 3.0%-39.4%)], and allo-HSCT markedly improved their 3-year OS [allo-HSCT: 47.6% (95% CI: 18.8%-100.0%) vs non-HSCT: 8.4% (95% CI: 1.3%-53.7%), P = 0.001]. In comparison, IKZF1plus/non-T315I subgroup showed a moderately better 3-year OS rate [42.9% (95% CI: 6.2%-79.5%), P = 0.17], with allo-HSCT associated with a trend toward improved prognosis. Lastly, IKZF1plus/no mutation subgroup demonstrated the highest 3-year OS rate [88.3% (95% CI: 78.5%-98.1%), P < 0.001], whose outcomes were similar in patients undergoing allo-HSCT or not. Additionally, transcriptomic profiling via RNA sequencing revealed that compared to IKZF1plus/no mutation subgroup,IKZF1plus/T315I subgroup was characterized by significant down-regulation of p53 signaling pathways (P = 0.03), indicating a potential molecular basis for their poor prognosis. Conclusion Integrating IKZF1plus genotyping and ABL1 mutation status, our findings firstly refine risk stratification and delineate distinct prognostic subgroups in high-risk adult BCR::ABL1+ ALL with IKZF1plusgenotype. Notably, IKZF1plus patients harboring T315I mutation constitute a subgroup with dismal survival, likely associated with the down-regulation of p53 pathways, and they would benefit from allo-HSCT. Conversely, IKZF1plus cases without ABL1 mutations exhibit a markedly favorable prognosis.

Monitoring NK-cell chimerism serves as a valuable addition to MRD-based surveillance.

Acute lymphoblastic leukemia (ALL) poses challenges in adult patients, considering its heterogeneous nature and often suboptimal treatment outcomes. Here, we performed a study on 201 newly diagnosed adult ALL cases (age ≥ 15 y) to generate intracellular and dynamic serum metabolomic profiles. Our findings revealed a predominant increase in bile acid (BA) metabolites in serum, alongside metabolic rewiring that supported highly proliferative states and actively metabolic signaling, such as enriched nucleotide metabolism in leukemic blasts. By integrating intracellular metabolomics and transcriptomics, we constructed the Comprehensive Metabolic Information Dataset (CMID), which facilitated the development of a clustering system to supplement current risk stratification. Furthermore, we explored potential metabolic interventions targeting the serum BA profile and energy metabolism in blasts. The combined use of simvastatin with vincristine and dexamethasone regimen demonstrated a synergistic therapeutic effect in a murine ALL model, effectively lowering key BA levels in serum and suppressing the infiltration of leukemic blasts in the liver. In light of the enhanced intracellular redox metabolism, combining FK866 (a nicotinamide phosphoribosyltransferase inhibitor) and venetoclax significantly prolonged survival in a patient-derived xenograft ALL model. Our findings, along with the resulting resources ( http://www.genetictargets.com/MALL ), provide a framework for the metabolism-centered management of ALL.

Flow cytometry (FCM), characterized by its simplicity, rapid processing, multiparameter analysis, and high sensitivity, is widely used in the diagnosis, treatment, and prognosis of hematological malignancies. FCM testing of tissue samples not only aids in diagnosing and classifying hematological cancers, but also enables the detection of solid tumors. Its ability to detect numerous marker parameters from small samples is particularly useful when dealing with limited cell quantities, such as in fine-needle biopsy samples. This attribute not only addresses the challenge posed by small sample sizes, but also boosts the sensitivity of tumor cell detection. The significance of FCM in clinical and pathological applications continues to grow. To standardize the use of FCM in detecting hematological malignant cells in tissue samples and to improve quality control during the detection process, experts from the Cell Analysis Professional Committee of the Chinese Society of Biotechnology jointly drafted and agreed upon this consensus. This consensus was formulated based on current literature and clinical practices of all experts across clinical, laboratory, and pathological fields in China. It outlines a comprehensive workflow of FCM-based assay for the detection of hematological malignancies in tissue samples, including report content, interpretation, quality control, and key considerations. Additionally, it provides recommendations on antibody panel designs and analytical approaches to enhancing FCM tests, particularly in cases with limited sample sizes.

DNA hypomethylating agents (HMAs) are widely used to treat acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS), but most treated patients relapse and lack standard treatment options. Using high-throughput screening, the approved all-trans retinoic acid (ATRA) is identified that exhibit high selectivity in killing HMA-resistant AML cells compared to parental cells. Mechanistically, HMA-resistant cells are overloaded with DNA hypomethylation-associated endogenous viral double-stranded RNA (dsRNA) which, however, fails to trigger an anticancer interferon (IFN) immune response due to downregulation of dsRNA sensor retinoic acid-inducible gene I (RIG-I). ATRA compensates for RIG-I expression, thereby re-triggering IFN response and potently inhibiting HMA-resistant AML cell lines, xenograft mice, and patient-derived primary cells. A library of potential RIG-I-inducing compounds is rationally constructed and screened, in which the approved M3 AML treatment drug tamibarotene (TAM) exhibits strikingly 28036-fold selectivity and 779 pm IC50 in killing HMA-resistant AML cells. ATRA and TAM do not selectively inhibit p53-mutant cancer cells. Together, this study uncovers a common resistance mechanism in HMA-treated AML patients and, in addition, provides highly potent and selective agents that can overcome resistance through re-triggering IFN anticancer immune response.

Immunomodulatory agent lenalidomide is effective in treating follicular lymphoma (FL). We conducted the first trial of immunotherapy rituximab plus lenalidomide in newly diagnosed FL in China (NCT03715309). One-hundred and fifteen patients were enrolled and treated with rituximab 375 mg/m2 intravenously on day 0 and lenalidomide 25 mg orally on day 1–10 for 6 cycles of induction treatment, as well as lenalidomide for 6 cycles and rituximab for 8 cycles of maintenance treatment. We found that inferior progression-free survival of the patients was significantly associated with elevated serum β2m and lymph node >6 cm, linking to decreased lymphoma cell autophagy and dendritic cell infiltration within the tumor microenvironment. PU.1 transcriptionally downregulated PD-L1 (Programmed death ligand 1) expression and upregulated 4-1BBL (4-1BB ligand) expression, increased lymphoma cell autophagy and dendritic cell maturation via PD-1/PD-L1 and 4-1BB/4-1BBL interaction. In vitro in co-culture system and in vivo in murine xenograft model, knockdown of PU.1 induced lenalidomide resistance, but sensitized FL cells to bi-specific PD-L1/4-1BB antibody or combined treatment of PD-L1 inhibitor and 4-1BB agonist. Collectively, PU.1 is essential in immunomodulatory effect of FL through PD-1/PD-L1- and 4-1BB/4-1BBL-mediated microenvironmental modulation. Dual targeting PD-L1 and 4-1BB could be an alternative immunotherapeutic strategy in the chemo-free era of FL treatment.

Introduction: Acute myeloid leukemia (AML) with t(16;21)(p11;q22)/FUS::ERG demonstrate poor outcomes even after allogeneic hematopoietic cell transplantation (allo-HCT). Up to now, most studies were from pediatric patients. Therefore, it is necessary to investigate the clinicopathological features, genomic and transcriptomic landscape, as well as treatment outcomes in a larger cohort to gain an in-depth understanding of adult patients. Methods: We initiated a retrospective study of FUS::ERG AML aged≥14 years in Chinese TROPHY group (Institutional Review Boards, No. 2025436). Patients diagnosed between May 2012 and May 2024 with complete medical records were enrolled. Patients were identified by reviewing karyotypes, PCR or RNA sequencing (RNA-Seq) results. The 2-year overall survival (OS), event free survival (EFS) and relapses were estimated using Kaplan–Meier curves and compared using log-rank test. The Prentice, Williams and Peterson total time (PWP-TT) model was used for analysis of recurrent time-to-event data, with results presented as hazard ratios (HR) and 95% confidence intervals (CI) for risk factors (considering age, gender, 2022 ELN risk stratification, and allo-HCT) regarding relapse. Data of 899 patients diagnosed in Ruijin Hospital with RNA-seq were provided as a reference cohort. Results: A total of 104 adult FUS::ERG AML were identified. Cytogenetic information was available in 101 patients, NGS were available in 64, and RNA-Seq data were available in 25. The median age was 35 (range: 14-73) years old, 13 patients aged older than 60 and 60 patients were male. Patients showed high WBC (median: 12.9×109/L) and bone marrow blast counts (median: 72%) at diagnosis. M5 (51%) was the predominant FAB subtype. Most leukemic blasts (80/82) were CD56 positive, and 31.2% (24/77) were classified as harboring RAM phenotype. Complex karyotypes were found in 41.3% of patients. PTPN11, RUNX1, and NRAS are the most prevalent somatic mutations. Ninety-two patients were stratified according to 2022 ELN recommendation with 56 (60.9%) cases in adverse-risk group. Ninety-one patients were eligible for intensive chemotherapy, 8 received low intensity induction, and 5 received supportive care. After two courses of induction, 83 (83.8%) patients achieved morphology complete remission (CR). The long-term survival of the entire cohort was dismal, with estimated 2-year probability of relapse, EFS, and OS of 77.2%, 18.7%, and 38.1%, respectively. Fifty-five patients underwent allo-HCT in first CR, 3 in second CR, and 7 with active disease. In CR1 cohort, both 2-year OS (allo-HCT: 59.1%, 95% CI: 46.2-75.5%; chemo: 5.1%, 95% CI: 0.8- 33.7%, P<0.0001) and EFS (allo-HCT: 34.1%, 95% CI: 22.7-51.2%; chemo: 0%, P<0.0001) were improved significantly with allo-HCT. The PWP-TT model revealed higher risk of relapse in patients aged≥60 years (HR: 8.1, 95% CI: 2.7–23.7, P<0.001), and allo-HCT can significantly reduce disease recurrence (HR: 6.5; 95% CI, 3.8–11.4, P<0.001). Compared to the reference cohort, FUS::ERG AML demonstrated similar long-term survival to that of the ELN adverse-risk group. Transcriptome-based analysis clustered all 25 cases together and classified into the myelodysplasia-related/-like subtype. Differential expression gene analysis revealed that insulin-like growth factor-2 mRNA-binding protein 1 (IGF2BP1) expression was significantly upregulated in FUS::ERG fusion AML comparing to reference cohort. Kyoto Encyclopedia of Genes and Genomes analysis showed enrichment of Rap1 and MAPK signaling pathway. Meanwhile, Gene Ontology analysis indicated that immune function-related pathways were significantly down-regulated, including immune system process, immune response, regulation of immune system, histocompatibility complex (MHC) protein complex binding, antigen binding and immune receptor activity. Elevated levels of IGF2BP1 correlated with loss of MHC class II expression (HLA-DR, -DQ, -DP, -DM, -DO) alongside suppression of most immune stimulators (CD28, CD80, CD86, KLRK1 and TNFSF/TNFRSF). Of note, the transcript level of TGFβ1 showed a significant elevation which is positively correlated with increase in IGF2BP1. Conclusions: We presented the largest comprehensive evidence to characterize a rare subtype of adult AML with FUS::ERG fusion which demonstrate poor outcomes with high relapse rate even after allo-HCT. Immune-evasion phenotype driven by IGF2BP1 overexpression may be the key factor contributing to the poor prognosis.

ABSTRACT:Acute myeloid leukemia (AML) is a highly heterogeneous hematological malignancy that increasingly affects the older population, with its posttranscriptional landscape remaining largely elusive. Establishing a stable proteomics-based classification system and systematically screening age-related proteins and regulatory networks are crucial for understanding the pathogenesis and outcomes of AML. In this study, we leveraged a multiomics cohort of 374 patients newly diagnosed with AML, integrating proteome, phosphoproteome, genome, transcriptome, and drug screening data. Through similarity network fusion clustering, we established 8 proteomic subtypes with distinct clinical and molecular properties, including S1 (CEBPA mutations), S3 (myelodysplasia-related AML), S4 (PML::RARA), S5 (NPM1 mutations), S6 (PML::RARA and RUNX1::RUNX1T1), S8 (CBFB::MYH11), S2 and S7 (mixed), aligning well with and adding actionable value to the latest World Health Organization nomenclature of AML. Hematopoietic lineage profiling of proteins indicated that megakaryocyte/platelet- and immune-related networks characterized distinct aging patterns in AML, which were consistent with our recent findings at the RNA level. Phosphosites also demonstrated distinct age-related features. The high protein abundance of megakaryocytic signatures was observed in S2, S3, and S7 subtypes, which were associated with advanced age and dismal prognosis of patients. A hematopoietic aging score with an independent prognostic value was established based on proteomic data, where higher scores correlated with myelodysplasia-related AML, NPM1 mutations, and clonal hematopoiesis-related gene mutations. Collectively, this study provides an overview of the molecular circuits and regulatory networks of AML during the aging process, advancing current classification systems and offering a comprehensive perspective on the disease.

BACKGROUND:CD19 chimeric antigen receptor (CAR) T-cell therapy is a potential treatment for relapsed/refractory (R/R) large B-cell lymphoma (LBCL). The combination of targeted therapeutic strategies, particularly bruton tyrosine kinase inhibitor zanubrutinib and programmed death-1 inhibitor tislelizumab, may improve clinical outcomes and modulate the tumour microenvironment (TME). METHODS:We studied patients with R/R LBCL who received response-adapted zanubrutinib plus tislelizumab upon CD19 CAR T-cell therapy between June 2021 and March 2023. Patients were treated with zanubrutinib daily from leukapheresis to day 28 post-infusion; those achieving complete response continued zanubrutinib monotherapy for 3 months, while partial responders received combined zanubrutinib for 3 months and tislelizumab for up to 2 years. We evaluated the overall response rate (ORR), complete response rate (CRR), progression-free survival (PFS), overall survival (OS), and safety. DNA sequencing and RNA sequencing were performed on available tumour samples to analyse genetic aberrations and TME characteristics. RESULTS:A total of 54 patients with LBCL were included, with a median follow-up of 23.6 months. The ORR at day 28, month 3, and month 6 were 94% (CRR 66%), 87% (CRR 80%), and 80% (CRR 76%), respectively. The 2-year PFS and 2-year OS rates were 68% and 76%, respectively. Median PFS and median OS were not reached. Grade ≥ 3 cytokine release syndrome occurred in 9% of patients, with no grade ≥ 3 neurotoxicity observed. Genomic and transcriptomic data indicated that this regimen was effective across genetic subtypes and abrogated T-cell exhaustion within the TME. However, tumour-infiltrating M2 macrophages with dysregulated lipid metabolism were associated with poor clinical outcome. CONCLUSIONS:Response-adapted zanubrutinib and tislelizumab potentially enhances the efficacy of CAR T-cell therapy with a favourable safety profile in R/R LBCL, effectively counteracting T-cell exhaustion. Future studies should focus on targeting M2 macrophages by reprogramming lipid metabolism to further attenuate the immunosuppressive TME. HIGHLIGHTS:Response-adapted zanubrutinib plus tislelizumab potentially enhances the efficacy of CAR T-cell therapy for R/R LBCL with acceptable safety profile. This regimen functions independently of genetic subtypes, rendering it more applicable for clinical practice with CAR T-cell therapy. This regimen effectively abrogates T-cell exhaustion, but fails to overcome the immunosuppressive effects of M2 macrophages, providing a rationale for remodelling TME to optimise CAR T-cell therapy.