Leukemia

We accrued 201 patients of adult AML treated with conventional therapy, in morphological remission and evaluated MRD using sensitive error corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients harbored PI NGS-MRD and 40.9% harbored PC NGS-MRD (median VAF 0.76%). Patients harboring NGS-MRD had a significantly higher cumulative incidence of relapse (p=0.003), inferior overall survival (p=0.001) and relapse free survival (p<0.001) as compared to NGS-MRD negative patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. Patients who cleared PI NGS-MRD had a significantly improved survival as compared to patients who became negative subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were truly missed by NGS as compared to FCM-MRD. NGS-MRD emerged as the most important independent prognostic factor predictive of inferior outcome (p<0.001). We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and advantageous when compared to FCM-MRD in AML treated with conventional therapies.

Little attention has been directed at untangling sex-related molecular and phenotypic differences in AML. While increased incidence and poor risk is generally associated with a male phenotype, FLT3-ITD, NPM1 and DNMT3A mutations are overrepresented in female AML. Here, we have investigated the relationship between sex and FLT3-ITD mutation status by comparing clinical data, mutational profiles, gene expression and ex vivo drug sensitivity responses in four cohorts: the Beat AML cohort, the LAML-TCGA cohort and two independent HOVON/SAKK clinical trial-associated cohorts, comprising a total of 1755 AML patients. We found that sex-associated molecular differences were prevalent in FLT3-ITD mutated AML. Co-occurrence of FLT3-ITD, NPM1 and DNMT3A mutations was overrepresented in females, while males with FLT3-ITDs were characterised by additional mutations in genes involved in RNA splicing and epigenetic modification. Female and male FLT3-ITD mutated AML had diverging expression of multiple leukemia-associated genes, as well as discrepant ex vivo drug-responses, suggestive of discrete functional properties. Surprisingly, we found significant prognostication of FLT3-ITD only in female patients. Thus, we suggest optimisation of FLT3-ITD mutation status as a clinical tool in a sex-adjusted manner. We further hypothesize that prognostication, prediction and development of therapeutic strategies in AML can be improved by including sex-specific considerations.

While outcome for pediatric T lymphoblastic lymphoma (T-LBL) has improved with Acute Leukemia-type therapy, survival after relapse remains rare. Few prognostic markers have been identified and the value of Minimal Residual Disease (MRD) is less clear than in T-ALL. Mutations of NOTCH1 and/or FBXW7 (N/F) identify good prognosis T-LBL and both MRD and high-level Minimal Disseminated Disease (MDD) are reported to be of poor prognosis. We evaluated MDD status by 8-color flow cytometry (MFC) and/or digital droplet PCR (ddPCR) in 86 French pediatric T-LBL, of which N/F status was known for 65 (61 treated on the Euro-LB02 protocol). Both techniques gave identical results for MDD/MRD values above 0.1%, allowing compilation. While an MDD threshold of 1% had no prognostic significance, the 54% (44/82) of protocol-treated patients with MDD [&ge;]0.1% had a relatively favorable outcome (overall survival/OS; p = 0.026). MDD 0.1% status had no prognostic significance in the 68% of patients with N/F mutations, whereas low/negative MDD status (9/61) identified N/F germline patients at a high risk of relapse (5-year OS of 44.4% vs 90% for MDD [&ge;] 0.1%,p = 0.014; and a 5-year DFS of 50% vs 90.9% respectively, p = 0.041). Combining oncogenetic and MDD status allows identification of 85% of patients with an excellent outcome (5-year OS 91.9% and DFS 95%) and 15% of N/F germline/MDD< 0.1% patients who clearly require early alternative treatment (5-year OS 44.4%; p< 0.0001 and DFS 50%; p = 0.0001).

Accurate and comprehensive genetic characterization of acute myeloid leukemia (AML) is essential for diagnosis, prognostication, and treatment selection. We report here, in 255 adults with AML enrolled in a prospective clinical protocol at 18 major cancer centers across the USA, the results of whole genome DNA-sequencing (WGS) at diagnosis and post-treatment remission. WGS effectively recapitulated, and frequently identified genetic alterations missed by, conventional standard of care clinical testing. These new findings included important prognostic and predictive biomarkers, copy number alterations, regulatory element, splicing, and structural variants including partial tandem duplications within KMT2A. All patients had a pathogenic variant detected at diagnosis, and approximately ten percent also had evidence of a potential inherited myeloid malignancy predisposition. This comprehensive atlas of adult AML genomics provides novel insights into disease biology, creates an evidentiary basis to support clinical testing improvements, and is a resource for both diagnostics and drug development. <This work is embargoed, with agreement of medRxiv, until 7th December 2025>. Statement of SignificanceAcute myeloid leukemia is a diagnostic category encompassing multiple rare hematological malignances. We show, in this nationwide multicenter study, that standardized unbiased whole genome DNA-sequencing and disease-optimized bioinformatics can replicate conventional "standard of care" AML clinical testing results, while also revealing currently underdiagnosed AML disease biology and potential genetic predisposition.

Nucleophosmin-1 (NPM1) mutations define a major molecular subtype of acute myeloid leukemia (AML) and is generally associated with favorable prognosis. However, the impact of myelodysplasia-associated mutations (MDSm+) on patient outcomes within this subgroup remains uncertain. We retrospectively analyzed 271 NPM1-mutated AML patients from three independent cohorts (SWOG, Fred Hutch, and Beat AML) to assess the prognostic significance of MDSm+ and its interaction with age. MDSm+ occurred in 17% of cases, most commonly involving SRSF2 and SF3B1. Although MDSm+ was associated with inferior overall survival compared to MDSm-in ELN2022 favorable-risk patients (HR 2.0, p=0.008), this effect was largely driven by worse outcomes in older patients ([&ge;]65 years) as older ELN22 favorable-risk patients had poor OS regardless of presence of MDSm+ compared to younger patients. After stratification of patients by age, there was not a significant difference between MDSm+ and MDSm-in either younger patients (HR 0.99, p=0.98) or older patients (HR 1.42, p=0.33). These findings indicate that MDSm+ in NPM1+ AML is not independently associated with adverse risk after adjusting for age and highlight the need for age-adjusted AML risk models.

WHO5-2022 classification of B-lymphoblastic leukemia (B-ALL) incorporates several novel entities requiring high-throughput sequencing for their accurate characterization. The clinical relevance of this classification in the context of contemporary MRD-directed therapy is unclear. We analyzed 533 pediatric B-ALL uniformly treated with ICiCLe-ALL-14 protocol as defined by WHO2016 and reclassified them as per WHO5-2022 using targeted sequencing, FISH, and cytogenetics. Subtype-defining genetic abnormalities were identified in 81.2% of the cohort as per the WHO5 classification. Among the new subtypes, PAX5alt, MEF2D-r, and BCR::ABL1-like(ABL-class) were associated with an inferior 2-year event-free survival (EFS) of 39.1% (p<0.0001), 53.8% (p=0.024) and 60.6% (p=0.043), respectively. We developed a 3-tier genetic risk stratification model incorporating 15 genetic subtypes and the IKZF1 deletion. Children with standard, intermediate, and high genetic risk demonstrated 2-year EFS of 92.6%, 71.0%, and 50.7% (p<0.0001), and 2-year overall survival of 94.3%, 81.9%, and 71.6% (p<0.0001), respectively. Genetic risk further identified heterogeneous outcomes among ICiCLe risk groups (p<0.0001). Standard genetic risk was associated with superior OS and EFS irrespective of MRD status. We demonstrate the applicability of the WHO5 classification in routine practice and create a general framework for incorporating the WHO5 classification in risk-adapted therapy for childhood B-ALL.

Occasional complete responses to immune checkpoint inhibitor therapies suggest that acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are immune-sensitive when appropriately targeted. Here, we analyzed AML/MDS patients treated with anti-TIM3 sabatolimab and decitabine in a phase Ib clinical trial (NCT03066648) using single-cell RNA and T cell receptor (TCR) sequencing and functional co-culture assays. Unlike T cell restricted CTLA4 and PD1, TIM3 was broadly expressed across natural killer (NK), myeloid, and T cell populations. Therapy induced expansion of cytotoxic NK cell subsets and enhanced type I interferon signaling. Less than 1% of bone marrow CD8+ T cells showed canonical exhaustion phenotypes, and the treatment preferably expanded small CD8+ T cell clones in responders. Responders had more cytotoxic CD4+ T and B cells which was exemplified by a patient with an outstanding complete response of 23-months. Over >20% of this patients lymphocytes were CD4+ T-cell large granular lymphocyte leukemia (T-LGLL) cells that expressed a TCR capable of recognizing autologous blasts. SignificanceAnti-TIM3+decitabine offers a distinct mechanism compared to anti-PD1 and anti-CTLA4: it preferentially enhances innate and unconventional cytotoxic lymphocyte populations. Furthermore, our findings suggest that clonal expansion of self-antigen-specific T cells, such as T-LGLL, may augment anti-leukemic immunity in the context of immunotherapy.

The presence of measurable residual disease (MRD) prior to an allogeneic hematopoietic transplant (alloHCT) in Acute Myeloid Leukemia (AML) has been shown to be associated with an increased risk of post-transplant relapse. Since the Isocitrate Dehydrogenase genes (IDH1/2) are mutated in a considerable proportion of patients with AML, we studied if these mutations would serve as useful targets for MRD. Fifty-five IDH-mutated AML patients undergoing non-myeloablative alloHCT with post-transplant cyclophosphamide at a single center were sequenced at baseline using a multi-gene panel followed by targeted testing for persistent IDH mutations at the pre- and post-alloHCT timepoints by digital droplet PCR or error-corrected next generation sequencing. The cohort included patients who had been treated with IDH inhibitors pre- and post-transplant (20% and 17% for IDH1 and 38% and 28% for IDH2). Overall, 55% of patients analyzed had detectable IDH mutations during complete remission prior to alloHCT. However, there were no statistically significant differences in overall survival (OS), relapse-free survival (RFS), and cumulative incidence of relapse (CIR) at 3 years between patients who tested positive or negative for a persistent IDH mutation during remission (OS: IDH1 p=1, IDH2 p=0.87; RFS: IDH1 p=0.71, IDH2 p= 0.78; CIR: IDH1 p=0.92, IDH2 p=0.97). There was also no difference in the prevalence of persistent IDH mutation between patients who did and did not receive an IDH inhibitor (p=0.59). Mutational profiling of available relapse samples showed that 8 out of 9 patients still exhibited the original IDH mutation, indicating that the IDH mutations remained stable through the course of the disease. This study demonstrates that persistent IDH mutations during remission is not associated with inferior clinical outcomes after alloHCT in patients with AML.

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukaemia remain largely unexplored. In this study we characterized mutations in SRSF2, U2AF1 and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline we accurately detected aberrant splicing in splicing factor mutated samples. Mutated samples were characterized predominantly by decreased junction usage. A large proportion of differentially used junctions were novel. Targets of splicing dysregulation included several genes with a known role in leukaemia. In SRSF2(P95H) mutants we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.

BackgroundCytogenetic analysis encompasses a suite of standard-of-care diagnostic testing methods that is routinely applied in cases of acute myeloid leukemia (AML) to assess chromosomal changes that are clinically relevant for risk classification and treatment decisions. ObjectiveIn this study, we assess the use of Genomic Proximity Mapping (GPM) for cytogenomic analysis of AML diagnostic specimens for detection of cytogenetic risk variants included in the European Leukemia Network (ELN) risk stratification guidelines. MethodsArchival patient samples (N=48) from the Fred Hutchinson Cancer Center leukemia bank with historical clinical cytogenetic data were processed for GPM and analyzed with the CytoTerra(R) cloud-based analysis platform. ResultsGPM showed 100% concordance for all specific variants that have associated impacts on risk stratification as defined by ELN 2022 criteria, and a 72% concordance rate when considering all variants reported by the FH cytogenetic lab. GPM identified 39 additional variants, including variants of known clinical impact, not observed by cytogenetics. ConclusionsGPM is an effective solution for the evaluation of known AML-associated risk variants and a source for biomarker discovery.

Acute Myeloid Leukemia (AML) is a genetically and clinically heterogeneous disease that can develop at any age. While AML incidence increases with age and distinct genetic alterations are observed in younger versus older patients, current classification systems do not incorporate age as a defining factor. In this study, we analyzed RNA-seq data from 404 AML patients at initial diagnosis, leveraging a k-mer-based machine learning approach to uncover age-related transcriptomic differences in favorable and adverse risk groups. Our model achieved over 90% accuracy in risk prediction and identified key gene signatures distinguishing ELN2017 favorable and adverse groups. From these signatures, we selected prognostic biomarkers with significant impacts on survival. Additionally, we explored the biological context underlying transcriptomic complexity across age groups, revealing distinct tumor profiles and differences in immune and stromal cell populations, particularly in older patients. These findings underscore the importance of age-related molecular features in AML and provide new insights for risk stratification and therapeutic targeting.

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

Accurate classification and risk stratification is critical for clinical decision making in AML patients. In the newly proposed World Health Organization (WHO) and International Consensus classifications (ICC) of hematolymphoid neoplasms, the presence of myelodysplasia-related (MR) gene mutations is included as one of the diagnostic criteria of AML, myelodysplasia-related (AML-MR), largely based on the assumption that these mutations are specific for AML with an antecedent myelodysplastic syndrome. ICC also prioritizes MR gene mutations over ontogeny (as defined by clinical history). Furthermore, European LeukemiaNet (ELN) 2022 stratifies these MR gene mutations to the adverse-risk group. By thoroughly annotating a cohort of 344 newly diagnosed AML patients treated at Memorial Sloan Kettering Cancer Center (MSKCC), we show that ontogeny assignment based on database registry lacks accuracy. MR gene mutations are frequently seen in de novo AML. Among MR gene mutations, only EZH2 and SF3B1 were associated with an inferior outcome in a univariate analysis. In a multivariate analysis, AML ontogeny had independent prognostic values even after adjusting for age, treatment, allo-transplant and genomic classes or ELN risks. Ontogeny also stratified the outcome of AML with MR gene mutations. Finally, de novo AML with MR gene mutations did not show an adverse outcome. In summary, our study emphasizes the importance of accurate ontogeny designation in clinical studies, demonstrates the independent prognostic value of AML ontogeny and questions the current classification and risk stratification of AML with MR gene mutations. Key pointsO_LIBoth ontogeny and genomics show independent prognostic values in AML. C_LIO_LIThe newly proposed myelodysplasia-related gene mutations are neither specific to AML-MRCWHO2016 nor predictive for adverse outcomes. C_LIO_LIOntogeny stratifies the outcome of AML with myelodysplasia-related gene mutations. C_LI

Recent genomic studies in adult and pediatric acute myeloid leukemia (AML) demonstrated recurrent in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF). These alterations, which account for ~4.3% of AMLs in childhood and up to 3% in adult AMLs under 60, are subtype-defining and associated with poor outcomes. Here, we provide a comprehensive investigation into the clinicopathological features of UBTF-TD myeloid neoplasms in childhood, including 89 unique pediatric AML and 6 myelodysplastic syndrome (MDS) cases harboring a tandem duplication in exon 13 of UBTF. We demonstrate that UBTF-TD myeloid tumors are associated with dysplastic features, low bone marrow blast infiltration, and low white blood cell count. Furthermore, using bulk and single-cell analyses, we confirm that UBTF-TD is an early and clonal event associated with a distinct transcriptional profile, whereas the acquisition of FLT3 or WT1 mutations is associated with more stem cell-like programs. Lastly, we report rare duplications within exon 9 of UBTF that phenocopy exon 13 duplications, expanding the spectrum of UBTF alterations in pediatric myeloid tumors. Collectively, we comprehensively characterize pediatric AML and MDS with UBTF-TD and highlight key clinical and pathologic features that distinguish this new entity from other molecular subtypes of AML. Key PointsO_LILargest cohort of pediatric UBTF-TD in myeloid neoplasms reported to date. C_LIO_LIUse of single-cell DNA+protein sequencing technology in 3 UBTF-TD samples reveals a clonal evaluation pattern characterized by sequential acquisition of WT1 and FLT3 mutations and a more stem cell-like protein expression pattern. C_LIO_LIPediatric MDS and AML patients with UBTF-TD alterations dysplastic features with an increase erythroid precursors. C_LIO_LITandem duplications in exon 9 of UBTF represent a rare but functionally equivalent subgroup of UBTF-TD myeloid neoplasms. C_LI Impact StatementUBTF tandem duplications (TD) are subtype-defining genomic alterations in adult and pediatric myeloid neoplasms. Here, we provide a comprehensive characterization of the largest cohort of pediatric UBTF-TD cases to date, including the recognition of additional UBTF alterations that mimic the exon 13 duplications in pediatric AML.

KMT2A partial tandem duplication (KMT2A-PTD) is a recurrent, high-risk alteration in myeloid neoplasms, yet no gold standard exists for its detection due to complex genomic architecture. We conducted a retrospective study of 97 specimens from 17 patients with KMT2A-PTD-positive myeloid neoplasms (11 AML, 4 MDS, 2 MPN) to compare chromosomal microarray analysis (CMA) and RNA fusion testing. Overall concordance was 73.3% ({kappa} = 0.467), with RNA fusion identifying more cryptic KMT2A-PTDs (p = 0.035), while CMA detected non-canonical KMT2A-PTDs and, additionally, secondary genomic abnormalities (e.g., trisomy 8 and CN-LOH of diverse genes). KMT2A-PTD cases exhibited high clonal complexity and distinct mutational profiles, including mutations of DNMT3A, TET2, ASXL1, RUNX1, FLT3, and others. Higher KMT2A-PTD transcript expression (split reads and SR/C ratio) correlated with DNMT3A, RUNX1, and FLT3 mutations, and RUNX1T1 copy number gain (p < 0.05), and was reduced in CN-LOH cases. RNA fusion consistently identified breakpoints at exon 10[-&gt;]2 or exon 11[-&gt;]2, affecting key oncogenic domains (CXXC, PHD finger, and bromodomain). Six-month overall survival was 41.2%, with HSCT significantly improving outcomes (68.6% vs. 0% at 17 months, p = 0.028), non-transplanted patients showing shorter median PFS ([~]6 months), and FLT3-mutated patients experiencing 100% mortality (p = 0.0456). In conclusion, CMA and RNA fusion analyses offer complementary diagnostic value in KMT2A-PTD-positive myeloid neoplasms. Such integrated genomic testing improves detection and refines risk stratification. Given the poor prognosis associated with KMT2A-PTD, especially in patients with co-occurring high-risk mutations, our findings support early consideration of HSCT in such patients.

BackgroundNatural killer (NK) cells originate from bone marrow precursors and mediate effective anti-tumor responses. Clinical trials of cytokine-primed memory-like (ML) NK cells in acute myeloid leukemia (AML) have demonstrated activity without major toxicity, including graft-versus-host disease or cytokine release syndrome. However, broad application of non-expanded, non-engineered ML NK cells has been hindered by limited availability of NK cells from a single donor, thereby precluding aggressive dose escalation and repeat dosing. W-NK1 is derived from human peripheral blood mononuclear cells undergoing ML reprogramming with a proprietary heteromeric fusion protein complex including IL-12, IL-15 and IL-18. MethodsWe conducted a multi-omics characterization of W-NK1 by interrogating its transcriptomic, proteomic and metabolic profile. Using functional assays, we assessed W-NK1s cytotoxicity under adverse culture conditions, as well as W-NK1s trafficking and killing abilities in immunodeficient mice engrafted with THP-1 AML. Finally, we evaluated W-NK1s phenotype and in vivo expansion kinetics in one patient with AML enrolled in study NCT05470140. ResultsW-NK1 displayed an activated, hyper-metabolic, and proliferative state differing from unstimulated conventional NK cells (cNK) from healthy donors. When compared to external single-cell NK datasets, W-NK1 was largely annotated as NKG2A+ and showed low relatedness with adaptive NK states characterized by HCMV-induced inflammatory memory. W-NK1 outperformed cNK cells in terms of in vitro killing of a broad panel of AML cell lines, with no appreciable cytotoxicity against normal cell lines. The expression of nutrient transporters was higher in W-NK1 compared to cNK cells and was retained even in adverse culture conditions designed to mimic an immunosuppressive tumor microenvironment. In mice engrafted with THP-1 AML, W-NK1 trafficked and efficiently homed to the bone marrow, where it mediated better tumor control than cNK cells. W-NK1 expanded, underwent phenotypic changes and persisted with effective elimination of circulating AML blasts through day 14 after infusion in one patient treated on clinical trial NCT05470140. Immunofluorescence staining of BM sections collected on day 28 showed increased expression of both CD56 and CD3 compared to a pre-treatment biopsy. ConclusionsOur study offers a comprehensive characterization of W-NK1 as an effective cell therapy product for AML and solid tumor malignancies. What is already known on this topicNatural killer (NK) cells have been shown to be safe and effective for treating certain human malignancies. Nonetheless, limitations for adoptive cell therapy exist which include trafficking / homing to tumor tissues as well as metabolic resilience in an adverse microenvironment. What this study addsW-NK1 is distinct transcriptionally and functionally from conventional NK cells with improved anti-tumor effector functions and metabolic adaptation in hostile culture conditions. Moreover, W-NK1 was readily detectable post-infusion in a patient with refractory acute myeloid leukemia. How this study might affect research, practice or policyOur in vitro and in vivo findings indicate that W-NK1 is an effective NK-cell therapy product and augur positively for patients being treated in phase I immunotherapy clinical trials.

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

Deleterious germline DDX41 variants are the leading cause of heritable predisposition to myelodysplastic syndrome 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 36 studies, including 1802 cases among 53795 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.

CD8+ T cells are critical players in anti-tumor immunity. In higher-risk myelodysplastic neoplasms (HR-MDS) and acute myeloid leukemia (AML), CD8+ T cells exhibit altered functionality, however whether this affects disease course is poorly understood. Herein, we aimed to identify immune cell signatures in the bone marrow (BM) associated with disease progression and treatment outcomes. In-depth immunophenotypic analysis utilizing mass and flow cytometry on 104 pre-treatment BM samples from patients with myeloid neoplasms, revealed an increased frequency of a CD57+CXCR3+ subset of CD8+ T cells in patients with HR-MDS and AML who failed AZA therapy. Furthermore, increased baseline frequency (>29%) of the CD57+CXCR3+CD8+ T cell subset correlated with poor overall survival. We further engaged scRNA-seq to assess the transcriptional profile of BM CD8+ T cells from treatment-naive patients. We observed an increased abundance of cells within cytotoxic CD8+ T lymphocytes (CTL) cluster in secondary AML compared to HR-MDS. Additionally, response to AZA was positively associated with enrichment of IFN-mediated pathways, whereas enhanced TGF-{beta} signaling signature in CTL clusters was observed in non-responders. Our results support that targeting of CD8+ T cells with inhibitors of TGF-{beta} signaling in combination with AZA is a potential future therapeutic strategy in HR-MDS and AML.

Causal germline genetic variants are frequently detected in young (under age 40) patients presenting with myelodysplastic syndromes (MDS) or bone marrow failure (BMF), where progression to acute myeloid leukemia (AML) contributes substantially to mortality in these patients. We reasoned that de novo pediatric AML, which shares clinical and biological characteristics, might also share germline genetic risk variants. We investigated germline variants in a large cohort (n=365) of pediatric AML patients with whole-genome sequencing (WGS), 29 with matched marrow-derived stromal cells, and 336 with matched remission marrow samples. Variants were deemed "likely germline" based on variant allele frequency (VAF) across available samples. Following American College of Medical Genetics and Genomics (ACMG) and Association of Molecular Pathology (AMP) guidelines, we annotated pathogenic/likely pathogenic (P/LP) variants in 555 genes linked to leukemia risk. P/LP variants were identified in 5.5% (95% CI: (3.3%,7.9%)) of patients in genes linked to familial myeloid malignancy and an additional 3.3% (95% CI: (1.6%,5.2%)) of patients in genes conferring risk to lymphoid malignancy or solid tumors. The large cohort enabled burden testing, which we employed by comparing loss-of-function variants between patients and 2504 control subjects from the 1000 Genomes Project. There was a 6.9-fold (95% CI: (3.1,14.9)) increase in loss-of-function variants in genes implicated in myeloid malignancy risk, a 2.4-fold (95% CI: (1.7,3.2)) increase in candidate risk genes, and a 1.6-fold (95% CI: (1.1,2.3)) increase in randomly-selected genes. We then assembled cohorts totaling 4,622 pediatric and adult patients with acute leukemia or MDS from 10 published studies, and compared P/LP variant burdens across age and diagnosis. The prevalence of germline variants in myeloid malignancies across age groups exceeds 5% consistently and with high confidence. Because the National Comprehensive Cancer Network recommends that all patients receive screening if their pre-test germline variant probability exceeds 5%, our results support germline genetic variant testing as an integral component of diagnostic work-up for myeloid malignancies, including donor selection for stem cell transplantation.