Novel staurosporine-type indolocarbazole glycoalkaloids as potent and selective FLT3-ITD inhibitors for acute myeloid leukemia.

Prognostic relevance of selected nucleotide variants in canine cutaneous mast cell tumors.

An internal tandem duplication in the gene encoding Fms-like tyrosine kinase 3 (FLT3-ITD) is associated with high relapse risk and poor prognosis in acute myeloid leukemia (AML) and plays a crucial role in treatment decisions. Measurable residual disease (MRD) analysis of FLT3-ITD during and after treatment has shown prognostic value in adults, but its clinical value in children remains unexplored. This study evaluated the potential of MRD analysis with deep sequencing (ultrahigh-sensitivity next-generation sequencing; UHS-NGS) of FLT3-ITD to assess treatment response and predict impending relapse in children with AML. A total of 168 samples from 17 patients (median age 11years, range: 4-17) were analyzed. Bone marrow samples were used for evaluation of treatment response, and peripheral blood samples for post-treatment monitoring. Deep sequencing enabled monitoring of the kinetics of FLT3-ITD subclones throughout treatment. When used for post-treatment monitoring, deep sequencing detected increasing levels of FLT3-ITD in peripheral blood only in patients who relapsed, while no such increase was detected in patients in continuous complete remission. Comparisons with flow cytometry and deep sequencing of other mutations confirmed the sensitivity of the assay and the often subclonal nature of FLT3-ITD. Deep sequencing of FLT3-ITD is a highly sensitive method for MRD monitoring in children with AML, with significant potential for assessing treatment response and detecting imminent relapse at an early stage. It may serve as a valuable molecular MRD method, especially as a complement to flow cytometry.

FLT3 inhibitors have improved outcomes in acute myeloid leukemia (AML) with FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD), but responses are not durable. Notably, FLT3 inhibitors clear blasts from the blood, but not the bone marrow, a hypoxic niche. We investigated effects of hypoxia and the key nutrient glutamine on FLT3 inhibitor response. FLT3-ITD AML cell lines and patient blasts were cultured with FLT3 inhibitors under normoxia (21%) or hypoxia (<1% O₂) with or without glutamine or the glutaminase inhibitor telaglenastat (CB-839). Cytotoxicity was measured in WST-1 assays and drug combination effects by Chou-Talalay analysis. Protein expression was measured by immunoblotting, turnover and proteasomal degradation by cycloheximide chase with and without MG-132, and mRNA expression by RT-qPCR. Effect of the ubiquitin ligase c-CBL was tested by siRNA knockdown. FLT3 inhibitor IC₅₀s were 3-5-fold higher in hypoxia than normoxia, associated with FLT3-ITD and p-STAT5 downregulation and accelerated FLT3-ITD proteasomal degradation (half-life, 1.0 vs. 2.5 hours). c-CBL expression increased in hypoxia, and c-CBL knockdown restored FLT3-ITD expression and FLT3 inhibitor sensitivity. Glutamine deprivation or telaglenastat treatment abrogated c-CBL upregulation in hypoxia and preserved FLT3-ITD and p-STAT5 expression and FLT3 inhibitor sensitivity. Telaglenastat synergized with FLT3 inhibitors in hypoxia, supporting clinical testing.

Despite advances, over half of AML patients relapse or become refractory to chemotherapy. TREM1 (CD354), a regulator of tumor inflammation and immune evasion in solid cancers, remains poorly characterized in AML, especially in underrepresented populations like Egyptians. We aimed to evaluate TREM1 expression in de novo AML, its association with high-risk features (FLT3-ITD, CD123), and its prognostic impact on treatment response and survival. In this prospective case-control study, TREM1 expression was quantified via flow cytometry (APC-conjugated anti-CD354) in 50 newly diagnosed AML patients and 50 matched controls. Patients were risk-stratified per ELN 2022 guidelines and treated with standard induction chemotherapy. TREM1 levels (CD45dim/SSC-low blasts) were correlated with molecular markers (FLT3-ITD, NPM1, cytogenetics), immunophenotypic profiles (CD2/CD7/CD19/CD56/CD123), and clinical outcomes (remission, survival). Statistical analyses included ROC-derived thresholds, Kaplan-Meier survival, and multivariate regression (SPSS v20.0). Key findings revealed significant TREM1 overexpression in AML blasts (median 21% [IQR: 12.8-43%]) compared to controls (15% [13-18%]; p = 0.01). Elevated TREM1 expression (> 21%) robustly predicted adverse prognosis (70% sensitivity, 75% specificity; AUC = 0.70, p = 0.04), chemotherapy resistance (non-complete remission: 77.8% sensitivity, 87% specificity; AUC = 0.80, p < 0.001), and inferior overall survival (median 5.5 vs. 11 months for low TREM1; p = 0.02). Notably, TREM1 expression correlated strongly with FLT3-ITD mutations (45.9% vs. 12.1% in wild-type; p = 0.01) and CD123 positivity (r = 0.29, p = 0.03), and was enriched in MLL-rearranged and complex karyotype subgroups (p = 0.04). Time-dependent ROC analysis confirmed sustained predictive accuracy (6-month AUC = 0.81), while TREM1/FLT3-ITD co-expression conferred a 5.4-fold mortality risk (p = 0.008). Subgroup analyses revealed consistent prognostic value across ELN risk categories. These results establish TREM1 as a biomarker of high-risk AML characterized by FLT3/CD123 co-expression, intrinsic chemotherapy resistance, and poor survival. The prognostic utility of TREM1 (cutoff > 21%) may enhance risk stratification, while its immunomodulatory function underscores its potential as a therapeutic target. Future studies should validate these findings in larger, multiethnic cohorts and explore the mechanistic links between TREM1 and AML pathogenesis.

Gilteritinib + chemotherapy in children with relapsed/refractory FLT3-ITD AML: results from the phase 1/2 SKIPPER trial.

Aberrant activation of TAL1 , a key oncogenic driver, defines a major subgroup comprising ∼30% of childhood T-lineage acute lymphoblastic leukemias (T-ALLs). We and others have shown that somatic non-coding mutations within upstream and intronic cis -regulatory regions of TAL1 contribute to transformation by creating binding sites for MYB and other transcription factors. Here we investigated cis -regulatory mechanisms mediated by somatic mutations occurring in an intergenic region located 29 kilobase pairs downstream of the canonical TAL1 transcription initiation site, implicated in 6% of TAL1 -expressing T-ALLs. These somatic variants include i) complex indels resulting in de novo MYB transcription factor binding sites (TFBSs) and ii) internal tandem duplications (ITDs) encompassing canonical MYB TFBSs. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed binding of the TAL1 core regulatory circuit (CRC) transcription factors MYB, GATA3, and RUNX1, resulting in enhancer activity mediated by sequences with the mutant allele. Strikingly, ChIP-seq peaks for the repressive H3K27me3 mark and the active H3K27ac mark co-existed across TAL1 regulatory sequences but enriched for different haplotypes. TAL1 transcription from the mutant haplotype initiated from a promoter located within exon 4 of the canonical TAL1 transcript, resulting in a short isoform normally expressed by hematopoietic stem cells (HSC). Interestingly, neither the isoform expression nor the enhancer activity could be predicted by the sequence-to-function deep learning artificial intelligence (AI) model AlphaGenome, emphasizing the importance of experimental validation. Our findings indicate that selection for cis -regulatory, non-coding variants leads to reactivation of enhancers normally active in HSC but silenced in differentiated lineages during normal hematopoietic cell development.

A 3-year-old female presented with a 1-week history of fevers and lethargy. Peripheral blood studies showed anaemia (haemoglobin: 79 g/L), thrombocytopenia (platelet count: 53× 109/L) and a normal leucocyte count (leucocyte count: 7.42 × 109/L) with 40% circulating blasts. Bone marrow evaluation revealed B-lymphoblastic leukaemia (B-ALL) without significant expression of myelomonocytic antigens by leukaemic blasts (Figure, top row left to right: blast morphology [Wright–Giemsa stain, objective x100 magnification] and representative flow cytometry dot plots). The B-lymphoblasts co-expressed CD371 (not shown), was indicative of an underlying DUX4 rearrangement (DUX4-r), which was subsequently confirmed by next-generation sequencing (NGS) analysis as a pathogenic IGH::DUX4 fusion.1 NGS also detected pathogenic variants of KRAS, NRAS, PTPN11 and an FLT3 internal tandem duplication mutation. Karyotyping revealed an isolated t(4;14)(q35;q32), and fluorescence in situ hybridization confirmed an IGH (14q32.33) rearrangement in 75% of cells. Following 8 days of standard induction chemotherapy, peripheral blood analysis (Figure, bottom row left to right: atypical monocyte morphology [Wright–Giemsa stain, objective ×100 magnification] and representative flow cytometry dot plots) identified residual B-lymphoblasts comprising 4% of cells (orange). In addition, a population of atypical monocytic cells (43%) was also detected (green) that expressed heterogeneous CD19, heterogeneous CD34 and co-expressed CD33, CD64 and CD14, consistent with a monocytic switch (MS) of leukaemic blasts. End of induction flow cytometric minimal residual disease evaluation of the bone marrow aspirate specimen demonstrated no evidence of residual atypical monocytes or B-lymphoblasts. The patient subsequently underwent consolidation therapy, including blinatumomab, without complications. Lineage infidelity and MS occurs in certain B-ALL subtypes, including those with mutated PAX5 P80R or rearrangements of DUX4, ZNF384 and CRLF2.2 In DUX4-r B-ALL, MS is a transient phenomenon occasionally observed during induction chemotherapy and is associated with an adverse event-free survival compared to DUX4-r B-ALL without MS. Recent single-cell ribonucleic acid sequencing study performed on DUX4-r leukaemic blasts demonstrated priming towards heterologous differentiation, including the monocytic and T-cell lineages in a subset of cases.3 Recognition of MS in the context of DUX4-r B-ALL during induction therapy is important to avoid its misdiagnosis as mixed phenotype acute leukaemia. Despite the slow initial response to induction chemotherapy and MS, DUX4-r B-ALL generally has a favourable outcome.4 The authors did not receive support from any organization for the submitted work. Retrospective review of pathology materials and clinical data was done with the approval by Cincinnati Children's Hospital Medical Center IRB. The authors have confirmed that a patient consent statement is not required for this submission, as no patient-identifying data were used. The authors declare no use of third-party material in this study for which formal permission is required.

This Phase I/II clinical trial (NCT04471064) evaluated the preliminary efficacy, safety, and pharmacokinetics of XY0206, a novel oral FMS-like tyrosine kinase 3 (FLT3) inhibitor, in patients with relapsed or refractory acute myeloid leukemia (R/R AML). From September 2020 to December 2022, this open-label, multicenter study enrolled patients aged ≥ 18 years with R/R AML. The trial included dose-escalation and dose-expansion phases, with six cohorts receiving XY0206 at doses ranging from 12.5 to 62.5 mg once daily or 25 mg twice daily. Of the 61 enrolled participants, 37 had FLT3 mutation-positive (FLT3mut+) AML. The overall response rate (ORR) was 34.4% in the entire cohort and 48.6% in FLT3mut+ patients. Among FLT3mut+ patients, the composite complete remission rate (CRc) was 45.9%, including a complete remission (CR) rate of 5.4% and a CR with partial hematologic recovery (CRh) rate of 13.5% and a CR with incomplete hematologic recovery (CRi) rate of 27.0%. In patients with FLT3 internal tandem duplication (FLT3-ITD) mutations, the ORR was 56.7%, with a CRc of 53.3% (CR: 6.7%; CRh: 16.7% ; CRi: 30.0%). The 37.5 mg dose cohort, identified as the target dose, was expanded exclusively for FLT3mut+ patients. XY0206 exhibited a favorable safety profile and demonstrated potent antileukemic activity, particularly in FLT3mut+ R/R AML patients, supporting its further clinical development. Trial Registration: CTR20201214 (CDE); ClinicalTrials.gov ID: NCT04471064.

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy. AML classification is currently based on cytogenetic and molecular alterations as well as immunophenotyping, although risk stratification still relies primarily on cytogenetic findings. However, approximately 45% of AML patients present with a normal karyotype, which makes accurate risk classification and treatment stratification more challenging. Therefore, the identification of molecular prognostic markers described in the literature has become essential in routine diagnostic laboratories, enabling the more precise categorization of patients into risk groups. In this study, we present a simple, rapid, step-by-step multiplex PCR protocol combined with capillary electrophoresis for the detection of two of the most prevalent molecular alterations in AML: nucleophosmin 1 (NPM1) mutations and Fms-like tyrosine kinase 3 internal tandem duplications (FLT3/ITD). This protocol provides a practical workflow that can assist diagnostic laboratories in implementing and optimizing multiplex mutation detection in routine practice.

Nintedanib is a potent FLT3 inhibitor with activity against FLT3-ITD and overcomes the gatekeeper F691L resistance mutation in acute myeloid leukemia.

HSR26-210: Patient Characteristics and Survival Outcomes in FLT3 Internal Tandem Duplication (ITD) vs. FLT3 Tyrosine Kinase Domain (TKD) Mutated AML: Insights from a Real-World Cohort.

To characterize the clinical, radiological, and molecular characteristics of CNS tumors associated with Noonan syndrome (NS) and other non-Neurofibromatosis type 1 RASopathies. Twenty-four patients with concern for NS underwent clinical and central radiological review in this multi-institutional study. Whole-exome sequencing, RNA sequencing, and methylation analyses of peripheral blood and/or tumor specimens were performed. Nineteen (79%) of 24 participants had NS, 17/19 (89%) of which had a germline PTPN11 variant; Nineteen of 24 participants (79%) were male. Seventeen of 19 (89%) patients with NS developed CNS tumors, including low-grade glioma, (LGG; pilocytic/pilomyxoid astrocytoma; n = 9) and dysembryoplastic neuroepithelial tumor (DNET; n = 6). Five patients incidentally diagnosed did not undergo histological confirmation. Radiological review showed multifocal parenchymal tumors in 9 patients with NS, including histologically confirmed neoplasm (n = 2), radiologic progression (n = 6), or typical tumoral imaging (n = 1). Fourteen of 15 (93%) tumors collected from 13 patients with NS and germline PTPN11 variants harbored somatic FGFR1 abnormalities. RNA sequencing of 12 tumors detected FGFR1 internal tandem duplication in one patient. Comparison with published data showed a statistically significant association between brain tumor occurrence and PTPN11-related NS, driven by two genotypes: NM_002834.5(PTPN11):c.182 A > G (p.Asp61Gly) and c.417G > T (p.Glu139Asp). Ten patients with CNS tumors, including 7/17 (41%) with PTPN11 variants, required chemotherapy. After median follow-up of 7.5 years, one patient died of CNS tumor. PTPN11-related NS predisposes to multifocal low-grade glial and glioneuronal tumors confirmed by radiological, histological, and molecular characteristics. Targeting FGFR1-related pathways may provide new treatment approaches for patients with NS and low-grade CNS tumors. Multifocal low-grade glial and glioneuronal tumors are common in patients with Noonan syndrome, especially in males. Concurrent PTPN11 and FGFR1 variants exist in nearly all patients with Noonan syndrome and low-grade CNS tumors. Targeting FGFR1 may provide novel treatment options for patients with Noonan syndrome and low-grade CNS tumors. Noonan syndrome (NS) has been recognized as a genetic predisposition to CNS tumors. However, no long-term clinical, radiological, or molecular patient data have previously been reported in a large patient cohort. Herein, we show that individuals with NS and germline PTPN11 variants, particularly those with NM_002834.5(PTPN11):c.182 A > G (p.Asp61Gly) and NM_002834.5(PTPN11):c.417G > C (p.Glu139Asp), are predisposed to develop low-grade tumors, including low-grade gliomas (LGGs) and dysembryoplastic neuroepithelial tumors (DNETs), which may have multifocal distant brain involvement in up to 50% of patients. An overwhelming male predominance was confirmed, for which the biological underpinnings remain unknown. Somatic FGFR1 variants were detected in 93% of tumors in individuals with PTPN11-related NS. Therefore, concurrent variants in both genes seem to be required to drive tumorigenesis. Most tumors exhibited indolent behavior, even in the presence of residual disease, and the affected patients experienced long-term survival. However, aggressive monitoring and treatment may be necessary in a subset of patients.

Acute Myeloid Leukemia (AML) is a genetically heterogeneous malignancy characterized by recurrent somatic mutations that influence disease biology and prognosis. Among these, internal tandem duplication (ITD) mutations in the FLT3 gene are among the most frequent and clinically adverse alterations. However, the prognostic relevance of ITD length and insertion site remains controversial, highlighting the need for further investigation. We conducted a single-center observational study including 384 newly diagnosed adult AML patients (pts) over 5 years, analyzed both prospectively and retrospectively. The FLT3-ITD mutation was detected in 72 pts (19%) by NGS or RT-PCR. ITD length (base pairs, bp) was correlated with clinical and biological features, and an optimal threshold was determined by Maximally Selected Rank Statistics (MAXSTAT). For 37 pts (51%) evaluated by NGS, the precise genomic insertion site was defined its biological and clinical relevance. The median age at diagnosis was 64 years. Co-mutations most frequently involved NPM1 (59%) and DNMT3A (43%). Multiple ITDs were detected in 6 pts. The median ITD length was 48 bp (IQR 30-69). Most insertions affected exon 14 (95%), while exon 15 and intronic regions were less commonly affected (5 and 16%, respectively). MAXSTAT analysis defined 63 bp as the optimal cut-point (Figure 1A), defining two distinct prognostic groups with comparable baseline features.Pts who relapsed showed significantly longer ITDs (median 71 vs 42 bp; p=0.018) (Figure 1B). A higher 5-year Cumulative Incidence of Relapse was observed in pts with long ITD (≥63 bp) (sHR=3.57; 95% CI:1.21–10.56; p=0.0216) (Figure 1C), with a consistent trend toward worse Overall and Event Free Survival. The negative prognostic effect of longer ITDs persisted after adjustment for age (p=0.0003), supporting ITD length as an independent predictor of outcome.Longer ITDs were more frequently associated with intronic involvement (p=0.001). No correlation was found between length and total number of co-mutations; however, DNMT3A-mutated pts displayed significantly longer ITDs and a fivefold higher likelihood of harboring ITDs ≥63 bp. Notably, pts harboring both DNMT3A mutation and long ITD (≥63 bp) (27%) had markedly reduced 5-year OS (p=0.02) (Figure 1D). Within the DNMT3A-mutated subgroup, longer ITDs also conferred inferior survival (p=0.03) (Figure 1E).Our findings demonstrate that longer FLT3-ITDs are associated with higher relapse risk and poorer clinical outcomes. We also identify a prognostically adverse interaction between DNMT3A mutation and ITD length. The preferential intronic involvement of long ITDs may indicate functional diversity with biological impact. These findings support integrating of ITD length, insertion site, and co-mutational status, particularly DNMT3A, into prognostic algorithms to improve risk stratification and guide treatment choice, including therapy intensification and indication for allogenic stem cells transplantation.

Myofibromas are part of the pericytic tumour family, which includes myopericytomas, glomus tumours and angioleiomyomas. While they typically display benign behaviour when arising in the skin and subcutaneous tissues of the head and neck, rare aggressive variants have been reported, particularly those with visceral or intracranial involvement. The most frequently identified molecular alterations in myofibromas are PDGFRB gain-of-function mutations, primarily single-nucleotide substitutions. Herein, we report two cases of myofibroma: one aggressive case with central nervous system involvement in a newborn, exhibiting a monophasic morphology, and a second, subcutaneous case in an adult. RNA sequencing was performed on both tumours, and data analysis was conducted using a four-pipeline fusion-calling approach (Arriba, FusionCatcher, FusionMap and STAR-Fusion). This analysis identified a novel somatic internal tandem duplication (ITD) in the NOTCH3 gene, affecting exons 26 and 27, specifically involving the C-terminal heterodimerization domain of the NOTCH3 receptor. Unsupervised hierarchical clustering demonstrated that myofibromas with ITDs segregate distinctly from conventional myofibromas and other pericytic tumours. Our findings suggest that NOTCH3 ITDs represent a novel oncogenic mechanism in pericytic tumour pathogenesis, likely driving constitutive activation of the NOTCH signalling pathway. Given the potential therapeutic relevance, particularly in aggressive or life-threatening cases with CNS involvement, our findings highlight the importance of extensive molecular profiling. Targeted therapy with NOTCH inhibitors may represent a promising strategy in the management of aggressive cases of ITD-driven pericytic tumours.

Patient-reported outcomes in newly diagnosed patients with FLT3-internal-tandem-duplication-positive acute myeloid leukaemia receiving standard chemotherapy plus quizartinib or placebo (QuANTUM-First): a global, randomised, placebo-controlled, phase 3 trial.

1278 Increased CD123 and Aberrant CD25 Expression are Common in Acute Myeloid Leukemia with KMT2A Partial Tandem Duplication and are Associated with FLT3 Internal Tandem Duplication

Design, synthesis, and Lead optimization of novel Quinazoline-based FLT3 inhibitors with potent anti-acute myelogenous leukemia activity.

Central Nervous System Metastases from Pediatric Solid Tumors: A Single-Institution Study.

Donor cell-derived myelodysplastic syndrome/acute myeloid leukemia is a rare but serious complication of allogeneic hematopoietic stem cell transplantation, and its optimal treatment has not been established. Here, we report a case of a 44-year-old woman who was diagnosed with donor cell-derived myelodysplastic syndromes with excess blasts carrying RAD21 and KMT2D mutations after six-year clinical remission of her initial acute myeloid leukemia treated with bone marrow transplantation from an unrelated male donor. The disease subsequently transformed to acute myeloid leukemia harboring a newly acquired FLT3 internal tandem duplication mutation. Azacitidine and venetoclax with cytarabine were both ineffective, but gilteritinib monotherapy rapidly reduced blasts and achieved near complete remission. The patient then underwent haploidentical stem cell transplantation from her son, followed by gilteritinib maintenance therapy. She achieved complete remission with clearance of donor cell-derived mutations and has remained in remission for more than one year. To our knowledge, this is the first case report of a relapsed FLT3-mutated donor cell-derived acute myeloid leukemia who was successfully treated with a FLT3 inhibitor, gilteritinib, which highlights the potential effectiveness of gilteritinib not only as a salvage therapy but also as an effective bridging and maintenance therapy in relapsed FLT3-mutated donor cell-derived acute myeloid leukemia.