Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Acute myeloid leukemia (AML) is a heterogeneous and aggressive hematologic malignancy with poor prognosis despite multiple available therapies. While chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment of B-cell malignancies, its application in AML has been limited by early relapses and severe toxicities. Unlike B-cell antigens, most AML-associated surface antigens are also expressed on healthy hematopoietic stem and progenitor cells, creating significant risks of on-target/off-tumor toxicity and prolonged myeloablation. To address the scarcity of AML-specific targets, several innovative CAR strategies have been developed to enhance precision, safety, and efficacy. Logic-gated CARs improve selectivity through dual-antigen recognition or conditional activation. Drug-inducible and transient expression systems, as well as pharmacologic or suicide switches, enable controlled modulation or elimination of CAR cells to reduce toxicity. Adapter CAR platforms allow real-time, flexible targeting, while engineered modulation of gene expression or cytokine secretion enhances persistence and antitumor activity. Finally, alternative immune cells, including natural killer (NK) cells and macrophages, provide versatile platforms that may overcome limitations of conventional T-cell therapies, such as fratricide or challenges in allogeneic use. This review provides a comprehensive overview of these emerging CAR approaches, highlighting their advantages, limitations, and potential to expand immunotherapeutic strategies for AML.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3′ splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1^mut/+), studied the U2af1^mut/+ hematopoietic system, and did not observe any gross differences in both young (12–13 weeks) and old (23 months) U2af1^mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1^mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1^mut/+Tet2^−/− mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1^mut/+Tet2^−/− mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.

Background/Objectives: In recent years, increasing evidence has supported CD84 as a promising therapeutic target in acute myeloid leukemia (AML), including its potential application in CAR T-cell therapy. CD84 is consistently reported to be highly expressed on AML cells in both adult and pediatric patients, providing a strong biological rationale for the development of CD84-directed immunotherapies. Methods: We characterized CD84 expression in bone marrow and peripheral blood samples from 61 adult patients with AML at diagnosis, relapse, and refractory disease using multiparametric flow cytometry. Results: Our results demonstrate that CD84 is highly expressed on AML cells throughout the course of the disease. More than 80% of leukemic populations showed CD84 expression above 80%, with median expression levels remaining consistently high at diagnosis (98%), relapse (96.5%), and refractory disease (96%). A novel aspect of our study is the evaluation of CD84 expression across distinct leukemic subpopulations within individual samples; only three of the 94 leukemic populations analyzed showed CD84 expression below 20%. Similarly, CD84 expression remained consistently high across the major AML subtypes analyzed, including AML with NPM1 mutation, AML with myelodysplasia-related gene mutations, and TP53-mutated AML. Comparable median CD84 expression was observed in bone marrow and peripheral blood; however, paired analysis identified significant differences between both compartments. CD84 expression assessed with antibody clone 153-4D9 was significantly lower than that detected with clone CD84.1.21 across AML leukemic populations (median, 75% vs. 98%; p < 0.0001). When compared with CD33 and CD123, CD84 showed a similarly broad prevalence of expression across AML leukemic populations. Healthy bone marrow samples were additionally evaluated to characterize the physiological distribution of CD84 expression across normal hematopoietic populations and provide a framework for the assessment of CD84-targeted therapeutic strategies. Conclusions: Together with the available preclinical and clinical evidence, these findings support the continued evaluation of CD84-directed immunotherapies, including CAR T-cell therapy, in AML.