Abstract FLT3-internal tandem duplication measurable residual disease testing for patients in remission from acute myeloid leukemia is now recommended by the recently updated clinical standard-of-care guidelines. This companion technical note provides important laboratory and clinical recommendations regarding such testing.

We report a novel in-frame CD274::PDCD1LG2 fusion in primary mediastinal B-cell lymphoma. Functional studies using expression constructs and CRISPR-engineered lymphoma cells confirmed formation of a PD-L1::PD-L2 chimeric protein and marked upregulation of PD-L1 surface expression, consistent with loss of 3′-UTR-mediated repression. These findings expand the genomic landscape of structural mechanisms driving PD-L1 activation and may inform variant interpretation and patient selection for PD-1 blockade in lymphoma.

Deleterious germline DDX41 variants are the leading cause of heritable predisposition to myelodysplastic neoplasia and acute myeloid leukemia (MDS/AML). Accurate classification of pathogenicity is crucial for managing patients and their families. The absence of specific guidelines, along with late-onset disease, incomplete penetrance, and founder variants, poses challenges in clinical and laboratory practice. We aggregated a synthetic cohort (ASC) of DDX41 germline and somatic variants from 35 studies, including 1796 cases among 53686 patients, plus an additional 832 cases from non-cohort publications. We aimed to leverage the DDX41-ASC to develop and refine ACMG/AMP criteria on case enrichment (PS4), somatic associations (PP4), and computational prediction (PP3/BP4). Analysis confirmed that deleterious germline DDX41 variants are most common in MDS/AML. A quasi-case-control study with ancestry matching revealed overestimated odds ratios for variants in underrepresented groups. Exploiting germline-somatic associations, we developed a Bayesian multinomial model that updates the odds of pathogenicity based on the presence and number of somatic patterns. Comparison of prediction tools showed that AlphaMissense outperformed REVEL in sensitivity. These results were integrated into an online tool to facilitate the consistent application of criteria. Overall, this comprehensive analysis of DDX41-ASC provides an evidence framework to inform the development of DDX41-specific curation guidelines.

Measurable residual disease (MRD) monitoring has become a critical component in the management of acute myeloid leukemia (AML), to inform prognosis, guide therapy, and serve as a key endpoint in clinical trials. The 2025 update of the MRD guideline provides a comprehensive and refined framework for MRD assessment, aligned with the ELN 2022 genetic risk classification. Developed by members of the ELN-DAVID consortium, the guidelines incorporate expert consensus determined through a two-stage Delphi round. They address the clinical implementation of MRD methodologies, technical considerations, integration into clinical trials, and future directions. Importantly, MRD recommendations are tailored to individual prognostic and genetic subgroups. A new qualitative MRD response category, designated as optimal, warning, or high risk of treatment failure, has been introduced to facilitate contextual interpretation of the MRD burden and its clinical relevance. Notably, ultrahigh-sensitivity (UHS) NGS-based MRD assessment is now recommended for FLT3-ITD-mutated AML following intensive chemotherapy and prior to allogeneic hematopoietic cell transplantation. A total of 56 recommendations were formulated, with 53 achieving a high level of consensus (≥90%). These updated guidelines represent a major step forward toward harmonizing MRD assessments in AML and enhancing its clinical utility across diverse treatment settings.

Genetic alterations activating NF‑κB have been described in mature B‑cell malignancies. These alterations include loss of the negative regulator TRAF3, allowing NIK (NF‑κB-inducing kinase) to drive non-canonical NF‑κB (ncNF‑κB) signaling. In this study, we characterized two recurrent synonymous variants of MAP3K14, the gene encoding NIK, across mature B‑cell malignancies including chronic lymphocytic leukemia, lymphoplasmacytic lymphoma/Waldenström's macroglobulinemia, multiple myeloma and mantle cell lymphoma. These variants were shown to introduce splice donor sites that result in removal of the critical TRAF3 binding motif. Cell line models prime edited to contain the synonymous variants demonstrated NF‑κB activation and transcriptional reprogramming with enhanced cell proliferation and survival, as well as increased secretion of several cytokines/chemokines including IP10/CXCL10. We also characterized distinctive cell surface proteomic changes with NF‑κB activation, identifying candidate markers (e.g.,CD132/IL‑2Rγ) for assessing NF‑κB activation at a single cell level using flow cytometry. This study expands the spectrum of genetic lesions that result in activation of NF‑κB in mature B‑cell malignancies and highlights our incomplete understanding of NF‑κB drivers in mature B‑cell malignancies.

Germline GATA2 deficiency is a pleiotropic condition 1-8 characterized by numerous phenotypes, including monocytopenia, immunodeficiency, microbial susceptibilities, and high rates of myeloid malignancies 1,8. Accurate curation of germline GATA2 variants is critical for patient care and requires well-defined phenotypes associated with GATA2 deficiency. The many phenotypes attributed to the condition render a simple description of GATA2 deficiency difficult, complicating the development of GATA2 variant curation rules. Therefore, the Myeloid Malignancy Variant Curation Expert Panel (MM-VCEP) sought to define GATA2 deficiency based on a statistical comparison of phenotype data. To do so, the MM-VCEP systematically analyzed phenotype data and applied statistical comparisons to define the phenotypic features of GATA2 deficiency to inform germline variant curation. The MM-VCEP assembled an international cohort of 339 people with clinically diagnosed GATA2 deficiency from 16 centers in seven countries. The 73 phenotypes of these individuals were compared statistically to those of control participants from the UK Biobank (UKBB). We compared single phenotypes as well as combinations of two, three, and four phenotypes in people with clinically diagnosed GATA2 deficiency to UKBB controls. We defined GATA2 deficiency as any of the 2,903 combinations of two or three phenotypes with log10Odds Ratio ≥3 (OR ≥ 1000). This definition of GATA2 deficiency will inform gene-specific phenotypic criteria used in GATA2 variant curation guidelines that will facilitate standardized variant curation by clinical laboratories worldwide.

ABSTRACT:Prognostic risk categorization aids treatment selection for patients with acute myeloid leukemia (AML). Although the European LeukemiaNet (ELN) classifications (2017 and 2022) for AML have been used to stratify outcomes for patients receiving intensive chemotherapy, their application to patients receiving less intensive therapy, such as azacitidine plus venetoclax, has been less satisfactory. In response, a 4-gene classifier that stratifies older patients with AML unfit for intensive chemotherapy into those with higher benefit (wild type), intermediate benefit (FLT3-internal tandem duplication [ITD] or NRAS/KRAS mutation), or lower benefit (TP53 mutation) after azacitidine plus venetoclax treatment was developed. We hypothesized that this 4-gene classifier may also have prognostic utility in patients receiving low-dose cytarabine (LDAC) plus venetoclax. Surprisingly, neither the ELN 2022 criteria nor the 4-gene azacitidine-venetoclax classifier model adequately stratified prognosis in a cohort of 139 patients receiving LDAC plus venetoclax. Patients with concurrent NPM1 and FLT3-ITD/RAS variants performed surprisingly well with LDAC plus venetoclax (complete remission [CR]/CR with incomplete blood count recovery [CRi] rate, 92%; median overall survival [OS], 29.67 months). Data-driven (sequential bootstrapping and tree-based) and empirical analyses identified complex karyotype and/or presence of TP53 mutation as prognostically relevant molecular/cytogenetic risk markers. Patients with complex karyotype and/or TP53 mutation displayed poor clinical outcomes (CR/CRi, 25%; median OS, 3.48 months). Notably, 74% of the study population lacked these poor prognostic markers and had a 67% CR/CRi rate with a median OS of 14.92 months. Overall, these data support the importance of molecular subclassification in defining treatment outcomes to venetoclax-based therapies. These trials were registered at www.clinicaltrials.gov as #NCT02287233 and #NCT03069352.

Venetoclax plus azacitidine represents a key advance for older, unfit patients with acute myeloid leukemia (AML). The chemotherapy and venetoclax in elderly AML trial (CAVEAT) was first to combine venetoclax with intensive chemotherapy in newly diagnosed patients ≥65 years. In this final analysis, 85 patients (median age 71 years) were followed for a median of 41.8 months. The CAVEAT induction combined cytarabine and idarubicin with 5 dose levels of venetoclax (50-600 mg) for up to 14 days. Two additional cohorts explored adjusted-dose venetoclax (50 mg, 100 mg) with posaconazole. CAVEAT induction was well tolerated, with low mortality (4%) and limited high-grade gastrointestinal toxicity (4%). Delayed hematological recovery after consolidation was ameliorated by omitting idarubicin from post-remission therapy. The overall response rate (ORR: CR + CRh + CRi) was 75% with a median overall survival (OS) of 19.3 months (95% CI 11.1-31.3). Among de novo AML, ORR was 88% and median OS 33.1 months (95% CI 19.3-54.3). Almost one-third have not relapsed, many benefiting from prolonged treatment-free remission (median 17.9 months). CAVEAT induction was well tolerated and associated with high ORR that was durable, particularly for de novo AML. CAVEAT represents an effective time-limited treatment option for fit older patients with AML. (https://www.anzctr.org.au; ACTRN12616000445471).

We describe two patients in whom malignant monoclonal T-cell lymphoproliferation developed after administration of chimeric antigen receptor (CAR) T-cell therapy with ciltacabtagene autoleucel (cilta-cel) in the phase 3 CARTITUDE-4 trial. Monoclonal T cells from both patients had detectable CAR transgene expression and integration. The clinicogenomic features of these CAR transgenic T-cell lymphoproliferative neoplasms suggest that multiple potential intrinsic or extrinsic factors (or both) contributed to their pathogenesis, such as transduction of preexisting TET2-mutated T cells, followed by acquisition of further oncogenic genomic variants. Other potential contributors include germline genomic variation, viral infections, and previous treatment for myeloma. In the absence of direct evidence, the contribution of insertional mutagenesis to the development of T-cell lymphoma is currently unclear. (Funded by Johnson & Johnson and Legend Biotech USA; CARTITUDE-4 ClinicalTrials.gov number, NCT04181827.).

Targeted RNA-based next-generation sequencing (targeted RNA-Seq) is increasingly incorporated into oncogenic gene fusion diagnostics. The absence of external quality assurance programs presents a challenge to the quality management of this novel diagnostic methodology. Four Australian molecular diagnostics laboratories employing Archer FusionPlex or Qiagen Fusion XP platforms compared results from six diagnostic samples with a reference method. Three of four laboratories identified all five gene fusions (BCR::ABL1, ETV6::RUNX1, ETV6::SRR, NUP98::HOXD13, KMT2A::USP2), and one laboratory missed the novel ETV6::SRR fusion. All laboratories missed the IGH::EPOR fusion identified by whole-transcriptome sequencing at a research laboratory; however, they were able to detect EPOR overexpression driven by the gene fusion. Diagnostic report comparison highlighted variations in the use of variant description nomenclature (Human Genome Variation Society vs International System for Human Cytogenomic Nomenclature), reference transcript, inclusion of genomic coordinates, and pathogenicity classification. This study demonstrates high concordance between laboratories in gene fusion identification using targeted RNA-Seq. Detecting non-chimeric gene producing rearrangements involving regulatory elements is a technical limitation of the current platforms. The incorporation of gene expression analysis, with confirmation using orthogonal methods, may help improve diagnostic yield. Developing practice guidelines will help harmonise reporting content and minimise interlaboratory variations.

Abstract The management of acute myeloid leukemia (AML) continues to evolve with ongoing improvements in risk stratification and the appropriate use of allogeneic transplantation. However, evidence of real-world AML survival improvements consistent with guideline adoption remains limited. We therefore evaluated survival outcomes and management of AML in younger adults aged ≤65 years across 2 decades (2002-2021) in an Australian statewide health network, contrasting differences between the more recent decade (2012-2021) and the preceding decade. A total of 329 patients were evaluated retrospectively using Kaplan-Meier methodology and multivariate Cox proportional hazards regression. Significantly, we observed an improvement in median overall survival between 2012 and 2021 compared with the 2002-2011 period (54 vs 22.2 months; P = .06). This was directly related to improvement in relapse-free survival (60 vs 21.3 months; P = .02) and the accompanying increased use of allogeneic transplantation (45% vs 28%; P = .003). Improvements were most profound in the intermediate cytogenetic risk group (median overall survival not reached vs 22 months; P = .02), with significant benefit observed for patients who underwent transplantation in this group, including those with FLT3-ITD mutations. Importantly, among patients with AML who did not undergo transplantation, we observed a lower relapse rate for those who had ≥3 consolidation cycles (58%) than those who had ≤2 (81%; P = .04). Notably, expert guidelines for transplantation were released and adopted by local hematologists immediately before this period, in 2010. Overall, our statewide registry data provide evidence of improved outcomes in AML, principally related to increased appropriate utilization of allogeneic transplantation and more granular risk assessment.

BACKGROUND:The preplanned interim analysis of the COMMANDS trial showed greater efficacy of luspatercept than epoetin alfa for treating anaemia in erythropoiesis-stimulating agent (ESA)-naive patients with transfusion-dependent, lower-risk myelodysplastic syndromes. In this Article, we report the results of the primary analysis of the trial. METHODS:COMMANDS is a phase 3, open-label, randomised, controlled trial conducted at 142 sites in 26 countries. Eligible patients were those aged 18 years or older, with myelodysplastic syndromes of very low risk, low risk, or intermediate risk (as defined by the Revised International Prognostic Scoring System), who were ESA-naive and transfusion dependent, and had a serum erythropoietin concentration of less than 500 U/L. Patients were stratified by baseline red blood cell transfusion burden, serum erythropoietin concentration, and ring sideroblast status, and randomly allocated (1:1) to receive luspatercept (1·0-1·75 mg/kg body weight, subcutaneously, once every 3 weeks) or epoetin alfa (450-1050 IU/kg body weight, subcutaneously, once a week; maximum total dose 80 000 IU) for at least 24 weeks. The primary endpoint was red blood cell transfusion independence lasting at least 12 weeks with a concurrent mean haemoglobin increase of at least 1·5 g/dL (weeks 1-24), evaluated in the intention-to-treat population. The safety population included all patients who received at least one dose of treatment. This trial is registered with ClinicalTrials.gov (NCT03682536; active, not recruiting). FINDINGS:Between Jan 2, 2019, and Sept 29, 2022, 363 patients were screened and randomly allocated: 182 (50%) to luspatercept and 181 (50%) to epoetin alfa. Median age was 74 years (IQR 69-80), 162 (45%) patients were female, and 201 (55%) were male. 289 (80%) were White, 44 (12%) were Asian, and two (1%) were Black or African American. 23 (6%) were Hispanic or Latino and 311 (86%) were not Hispanic or Latino. Median follow-up for the primary endpoint was 17·2 months (10·4-27·7) for the luspatercept group and 16·9 months (10·1-26·6) for the epoetin alfa group. A significantly greater proportion of patients in the luspatercept group reached the primary endpoint (110 [60%] vs 63 [35%]; common risk difference on response rate 25·4% [95% CI 15·8-35·0]; p<0·0001). Median follow-up for safety analyses was 21·4 months (IQR 14·2-32·4) for the luspatercept group and 20·3 months (12·7-30·9) for the epoetin alfa group. Common grade 3-4 treatment-emergent adverse events occurring among luspatercept recipients (n=182) were hypertension (19 [10%] patients), anaemia (18 [10%]), pneumonia (ten [5%]), syncope (ten [5%]), neutropenia (nine [5%]), thrombocytopenia (eight [4%]), dyspnoea (eight [4%]), and myelodysplastic syndromes (six [3%]); and among epoetin alfa recipients (n=179) were anaemia (14 [8%]), pneumonia (14 [8%]), neutropenia (11 [6%]), myelodysplastic syndromes (ten [6%]), hypertension (eight [4%]), iron overload (seven [4%]), and COVID-19 pneumonia (six [3%]). The most common serious treatment-emergent adverse events in both groups were pneumonia (nine [5%] luspatercept recipients and 13 [7%] epoetin alfa recipients) and COVID-19 (eight [4%] luspatercept recipients and ten [6%] epoetin alfa recipients). One death (due to acute myeloid leukaemia) considered to be luspatercept-related was reported at the interim analysis. INTERPRETATION:Luspatercept represents a new standard of care for ESA-naive patients with transfusion-dependent, lower-risk myelodysplastic syndromes. Significantly more patients had red blood cell transfusion independence and haematological improvement with luspatercept than with epoetin alfa, with benefits observed across patient subgroups. FUNDING:Celgene and Acceleron Pharma.

The classification of myeloid neoplasms continues to evolve along with advances in molecular diagnosis, risk stratification and treatment of disease. An approach for disease classification has been grounded in international consensus that has facilitated understanding, identification and management of molecularly heterogeneous entities, as well as enabled consistent patient stratification into clinical trials and clinical registries over time. The new World Health Organization (WHO) and International Consensus Classification (ICC) Clinical Advisory Committee releasing separate classification systems for myeloid neoplasms in 2022 precipitated some concern amongst haematopathology colleagues both locally and internationally. While both classifications emphasise molecular disease classification over the historical use of morphology, flow cytometry and cytogenetic based diagnostic methods, notable differences exist in how morphological, molecular and cytogenetic criteria are applied for defining myelodysplastic neoplasms (MDS) and acute myeloid leukaemias (AML). Here we review the conceptual advances, diagnostic nuances, and molecular platforms required for the diagnosis of MDS and AML using the new WHO and ICC 2022 classifications. We provide consensus recommendations for reporting bone marrow biopsies. Additionally, we address the logistical challenges encountered implementing these changes into routine laboratory practice in alignment with the National Pathology Accreditation Advisory Council reporting requirements for Australia and New Zealand.

Despite an increasing desire to use historical cohorts as “synthetic” controls for new drug evaluation, limited data exist regarding the comparability of real-world outcomes to those in clinical trials. Governmental cancer data often lacks details on treatment, response, and molecular characterization of disease sub-groups. The Australasian Leukaemia and Lymphoma Group National Blood Cancer Registry (ALLG NBCR) includes source information on morphology, cytogenetics, flow cytometry, and molecular features linked to treatment received (including transplantation), response to treatment, relapse, and survival outcome. Using data from 942 AML patients enrolled between 2012–2018, we assessed age and disease-matched control and interventional populations from published randomized trials that led to the registration of midostaurin, gemtuzumab ozogamicin, CPX-351, oral azacitidine, and venetoclax. Our analyses highlight important differences in real-world outcomes compared to clinical trial populations, including variations in anthracycline type, cytarabine intensity and scheduling during consolidation, and the frequency of allogeneic hematopoietic cell transplantation in first remission. Although real-world outcomes were comparable to some published studies, notable differences were apparent in others. If historical datasets were used to assess the impact of novel therapies, this work underscores the need to assess diverse datasets to enable geographic differences in treatment outcomes to be accounted for.