Internal Tandem Duplication Research Articles

Quizartinib: a potent and selective FLT3 inhibitor for the treatment of patients with FLT3-ITD-positive AML.

Mutations in FMS-related receptor tyrosine kinase 3 (FLT3) are among the most common alterations in acute myeloid leukemia (AML), present in ≈30% of newly diagnosed AML cases. Internal tandem duplications (ITD) in FLT3 (FLT3-ITD) occur in ≈25% of newly diagnosed AML cases and are associated with unfavorable outcomes. Quizartinib (formerly AC220) is a novel, second-generation, highly potent, and selective type II FLT3 inhibitor. Quizartinib is approved in Japan as monotherapy for the treatment of adult patients with FLT3-ITD-positive relapsed/refractory (R/R) AML. Quizartinib is also approved in the United States, Japan, Europe, and United Kingdom in combination with chemotherapy during induction and consolidation, and as maintenance monotherapy (but, in the United States, not after allogeneic hematopoietic cell transplantation [allo-HCT]), for the treatment of adult patients with newly diagnosed FLT3-ITD-positive AML. In this review, we summarize preclinical studies that established quizartinib as a potent and selective type II FLT3 inhibitor as well as early and pivotal phase 3 clinical studies (QuANTUM-R and QuANTUM-First) that led to the approvals of quizartinib. We also summarize mechanisms of resistance to quizartinib along with its safety profile. Furthermore, we review the ongoing post hoc analyses of the QuANTUM-First data elucidating the impact of allo-HCT, the presence of measurable residual disease, and number and length of ITD on the clinical outcomes of quizartinib. We also describe the impact of quizartinib on patient-reported outcomes. Finally, we highlight some of the ongoing studies that test quizartinib in patients with FLT3-ITD-positive AML, patients with FLT3-ITD-negative AML, in both the first-line and R/R settings, in patients fit or unfit for intensive chemotherapy, including studies for quizartinib-based combination with other compounds such as decitabine and venetoclax. Future research should aim to further optimize the clinical value of quizartinib and explore its use in additional clinical settings, which could be achieved by testing quizartinib with other drugs, better characterization of the mechanisms of resistance, identification of the role of quizartinib as a maintenance therapy after allo-HCT, and investigating quizartinib in patients with FLT3-ITD-negative AML.

BIOM-51. MOLECULAR SUBTYPES, CLINICAL CHARACTERISTICS, AND THERAPEUTIC IMPLICATIONS FOR CNS TUMORS WITHBCOR/BCORL1 FUSION/INTERNAL TANDEM DUPLICATION

Abstract Central nervous system (CNS) tumors with either BCOR internal tandem duplication (BCOR ITD) or gene fusions involving BCOR/BCORL1 (BCOR FUS) are recognized as tumor types with characteristic molecular and clinical features but lacking clear therapeutic guidance. In an extensive international effort, we compiled a retrospective cohort of 229 (148 BCOR ITD, 81 BCOR FUS) unique cases via molecular profiling of CNS tumors. By analyzing DNA methylation patterns of 220 tumors we confirmed distinct epigenetic profiles of BCOR ITD versus BCOR FUS. The majority (91 percent) of BCOR FUS primary tumors was localized in the supratentorial region whereas BCOR ITD were localized across all anatomical CNS compartments. Median patient age at diagnosis was 20 years for BCOR FUS and 4 years for BCOR ITD. Metastatic disease was rare in BCOR FUS patients but more common in BCOR ITD tumors where 10 percent presented with metastatic disease and one third showed CNS or (rarely) extra-CNS metastasis in the course of disease. This was also reflected in clinical outcome as 5-year overall survival (OS) was 89 percent in BCOR FUS but 61 percent in BCOR ITD. In contrast, 5-year progression-free survival (PFS) was comparable reaching 27 percent in BCOR FUS and 30 percent in BCOR ITD. Integrating treatment details we found a trend towards benefit of gross-total resection on OS (BCOR FUS, n=18, p<0.08; BCOR ITD, n=67, p<0.12) but not for PFS (BCOR FUS, n=16, p<0.33; BCOR ITD, n=64, p<0.59). Radiotherapy was associated with improved PFS (BCOR FUS, n=18, p<0.01; BCOR ITD, n=70, p<0.001) and OS (BCOR FUS, n=20, p<0.01; BCOR ITD, n=73, p<0.001). Further in-depth analyses of clinical outcome parameters (primary metastatic disease, radiotherapy approach, chemotherapy type, patient age) are currently ongoing. In summary, we provide first robust annotation of CNS BCOR tumor types, clinical outcomes and therapy.

PRDM16 Induces Methylation of FLT3 to Promote FLT3-ITD Signaling and Leukemia Progression.

Internal tandem duplication (ITD) in the FMS-like receptor tyrosine kinase-3 (FLT3) is one of the most frequent mutations in acute myeloid leukemia (AML) and is associated with poor prognosis. FLT3-ITD mutations result in endoplasmic reticulum (ER) retention and constitutive autophosphorylation of FLT3. The PR/SET domain 16 (PRDM16) is highly expressed in FLT3-ITD+ AML patients, suggesting it might play a role in leukemogenesis. Here, we revealed that genetic and pharmacological suppression of PRDM16 greatly slowed the progression of FLT3-ITD-driven leukemia, sensitized leukemic cells to tyrosine kinase inhibitors (TKIs), and extended the survival of leukemic mice. PRDM16 enhanced activation of oncogenic FLT3-ITD and ligand-dependent activation of wild-type FLT3 in leukemic cells. Mechanistically, PRDM16 mediated monomethylation of FLT3-ITD at lysine 614 and promoted its ER localization, resulting in enhanced FLT3 signaling in leukemia cells. Moreover, pharmacological suppression of FLT3-ITD methylation in combination with TKIs increased the elimination of FLT3-ITD+ AML cells. Altogether, these results suggest that PRDM16 boosts oncogenic FLT3 signaling in leukemic cells by prompting FLT3-ITD methylation. Therefore, PRDM16 may serve as a therapeutic target for AML.

The Immunomodulatory Effect of Different FLT3 Inhibitors on Dendritic Cells.

FMS-like tyrosine kinase 3 (FLT3) mutations or internal tandem duplication occur in 30% of acute myeloid leukemia (AML) cases. In these cases, FLT3 inhibitors (FLT3i) are approved for induction treatment and relapse. Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains the recommended post-induction therapy for suitable patients. However, the role of FLT3i therapy after alloHSCT remains unclear. Therefore, we investigated the three currently available FLT3i, gilteritinib, midostaurin, and quizartinib, in terms of their immunosuppressive effect on dendritic cells (DCs). DCs are professional antigen-presenting cells inducing T-cell responses to infectious stimuli. Highly activated DCs can also cause complications after alloHSCT, such as triggering Graft versus Host disease, a serious and potentially life-threatening complication after alloHSCT. To study the immunomodulatory effects on DCs, we differentiated murine and human DCs in the presence of FLT3i and performed immunophenotyping by flow cytometry and cytokine measurements and investigated gene and protein expression. We detected a dose-dependent immunosuppressive effect of midostaurin, which decreased the expression of costimulatory markers like CD86, and found a reduced secretion of pro-inflammatory cytokines such as IL-12, TNFα, and IL-6. Mechanistically, we show that midostaurin inhibits TLR and TNF signaling and NFκB, PI3K, and MAPK pathways. The immunosuppressive effect of gilteritinib was less pronounced, while quizartinib did not show truncation of relevant signaling pathways. Our results suggest different immunosuppressive effects of these three FLT3i and may, therefore, provide an additional rationale for optimal maintenance therapy after alloHSCT of FLT3-positive AML patients to prevent infectious complications and GvHD mediated by DCs.

SARCP, A Clinical Next Generation Sequencing Assay for the Detection of Gene Fusions in Sarcomas: A Description of the First 652 Cases

An amplicon-based targeted next generation sequencing (NGS) assay for the detection of gene fusions in sarcomas was developed, validated, and implemented. This assay can detect fusions in targeted regions of 138 genes and BCOR internal tandem duplications. This study reviews our experience with testing on the first 652 patients analyzed. Gene fusions were detected in 238 of 652 (36.5%) of cases, including 83 distinct fusions in the 238 fusion positive cases, 10 of which had not been previously described. Among the 238 fusion positive cases, the results assisted in establishing a diagnosis for 137 (58%) cases, confirmed a suspected diagnosis in 66 (28%) cases, changed a suspected diagnosis in 25 (10%) cases and were novel fusions with unknown clinical significance in 10 (4%) cases. Twenty-six cases had gene fusions (ALK, ROS1, NTRK1, NTRK3, and COL1A1::PDGFB) for which there are targetable therapies. BCOR ITDs were identified in 6 of 485 (1.2%) patients. Among the 138 genes in the panel, 66 were involved in one or more fusions and 72 were not involved in any fusions. There was little overlap between the genes involved as 5’-partners (31 different genes) and 3’-partners (37 different genes). This study demonstrates the clinical utility of a next generation sequencing gene fusion detection assay for the diagnosis and treatment of sarcomas.

Artificial intelligence can be trained to predict c-KIT-11 mutational status of canine mast cell tumors from hematoxylin and eosin-stained histological slides.

Numerous prognostic factors are currently assessed histologically and immunohistochemically in canine mast cell tumors (MCTs) to evaluate clinical behavior. In addition, polymerase chain reaction (PCR) is often performed to detect internal tandem duplication (ITD) mutations in exon 11 of the c-KIT gene (c-KIT-11-ITD) to predict the therapeutic response to tyrosine kinase inhibitors. This project aimed at training deep learning models (DLMs) to identify MCTs with c-KIT-11-ITD solely based on morphology. Hematoxylin and eosin (HE) stained slides of 368 cutaneous, subcutaneous, and mucocutaneous MCTs (195 with ITD and 173 without) were stained consecutively in 2 different laboratories and scanned with 3 different slide scanners. This resulted in 6 data sets (stain-scanner variations representing diagnostic institutions) of whole-slide images. DLMs were trained with single and mixed data sets and their performances were assessed under stain-scanner variations (domain shifts). The DLM correctly classified HE slides according to their c-KIT-11-ITD status in up to 87% of cases with a 0.90 sensitivity and a 0.83 specificity. A relevant performance drop could be observed when the stain-scanner combination of training and test data set differed. Multi-institutional data sets improved the average accuracy but did not reach the maximum accuracy of algorithms trained and tested on the same stain-scanner variant (ie, intra-institutional). In summary, DLM-based morphological examination can predict c-KIT-11-ITD with high accuracy in canine MCTs in HE slides. However, staining protocol and scanner type influence accuracy. Larger data sets of scans from different laboratories and scanners may lead to more robust DLMs to identify c-KIT mutations in HE slides.

Midostaurin shapes macroclonal and microclonal evolution of FLT3-mutated acute myeloid leukemia

AbstractDespite the use of midostaurin (MIDO) with intensive chemotherapy (ICT) as the frontline treatment for Fms-like tyrosine kinase 3 (FLT3)-mutated acute myeloid leukemia (AML), complete remission rates are close to 60% to 70%, and relapses occur in >40% of cases. Here, we studied the molecular mechanisms underlying refractory/relapsed (R/R) disease in patients with FLT3-mutated AML. We conducted a retrospective and multicenter study involving 150 patients with R/R AML harboring FLT3–internal tandem duplication (ITD) (n = 130) and/or FLT3–tyrosine kinase domain (n = 26) at diagnosis assessed by standard methods. Patients were treated in front line with ICT + MIDO (n = 54) or ICT alone (n = 96) according to the diagnosis date and label of MIDO. The evolution of FLT3 clones and comutations was analyzed in paired diagnosis–R/R samples by targeted high-throughput sequencing. Using a dedicated algorithm for FLT3-ITD detection, 189 FLT3-ITD microclones (allelic ratio [AR] of <0.05) and 225 macroclones (AR ≥ 0.05) were detected at both time points. At R/R disease, the rate of FLT3-ITD persistence was lower in patients treated with ICT + MIDO than in patients not receiving MIDO (68% vs 87.5%; P = .011). In patients receiving ICT + MIDO, detection of multiple FLT3-ITD clones (referred to as “clonal interference”) was associated with a higher FLT3-ITD persistence rate at R/R disease (multiple clones: 88% vs single clones: 57%; P = .049). Considering both treatment groups, if only 24% of FLT3-ITD microclones detected at diagnosis were retained at relapse, 43% became macroclones. Together, these results identify parameters influencing the fitness of FLT3-ITD clones and highlight the importance of using sensitive techniques for FLT3-ITD screening in clinical practice.

Targeting rapid TKI-induced AXL upregulation overcomes adaptive ERK reactivation and exerts antileukemic effects in FLT3/ITD acute myeloid leukemia.

Acute myeloid leukemia (AML) patients with the FMS-related receptor tyrosine kinase 3 internal tandem duplication (FLT3/ITD) mutation have a poorer prognosis, and treatment with FLT3 tyrosine kinase inhibitors (TKIs) has been hindered by resistance mechanisms. One such mechanism is known as adaptive resistance, in which downstream signaling pathways are reactivated after initial inhibition. Past work has shown that FLT3/ITD cells undergo adaptive resistance through the reactivation of extracellular signal-regulated kinase (ERK) signaling within 24 h of sustained FLT3 inhibition. We investigated the mechanism(s) responsible for this ERK reactivation and hypothesized that targeting tyrosine-protein kinase receptor UFO (AXL), another receptor tyrosine kinase that has been implicated in cancer resistance, may overcome the adaptive ERK reactivation. Experiments revealed that AXL is upregulated and activated in FLT3/ITD cell lines mere hours after commencing TKI treatment. AXL inhibition combined with FLT3 inhibition to decrease the ERK signal rebound and to exert greater anti-leukemia effects than with either treatment alone. Finally, we observed that TKI-induced AXL upregulation occurs in patient samples, and combined inhibition of both AXL and FLT3 increased efficacy in our in vivo models. Taken together, these data suggest that AXL plays a role in adaptive resistance in FLT3/ITD AML and that combined AXL and FLT3 inhibition might improve FLT3/ITD AML patient outcomes.

Measurable residual disease monitoring in AML with FLT3-ITD treated with intensive chemotherapy plus midostaurin

AbstractMeasurable residual disease (MRD) monitoring in acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITDpos]) has been hampered by the broad heterogeneity of ITD mutations. Using our recently developed FLT3-ITD paired-end next-generation sequencing (NGS)–based MRD assay with a limit of detection of 10−4 to 10−5, we evaluated the prognostic impact of MRD at different time points in 157 patients with FLT3-ITDpos AML who were enrolled in the AMLSG16-10 trial and who were treated with a combination of intensive chemotherapy and midostaurin, followed by midostaurin maintenance. Achievement of MRD negativity (MRDneg) after 2 cycles of chemotherapy (Cy2), which was observed in 111 of 142 (78%) patients, was predictive of superior 4-year rates of cumulative incidence of relapse (CIR) (4y-CIR; 26% vs 46%; P = .001) and overall survival (OS) (4y-OS; 70% vs 44%; P = .012). This survival advantage was also seen among patients who underwent allogeneic hematopoietic-cell transplantation during first complete remission (4y-CIR, 14% vs 39%; P = .001; 4y-OS, 71% vs 49%; P = .029). Multivariate models for CIR and OS after Cy2 revealed FLT3-ITD MRDneg as the only consistent favorable variable for CIR (hazard ratio [HR], 0.29; P = .006) and OS (HR, 0.39; P = .018). An NPM1 co-mutation correlated with a deeper molecular response as reflected by stronger MRD reduction and a higher rate of FLT3-ITD MRDneg after Cy2. During follow-up, conversion from MRDneg to MRDpos was a strong, independent factor for inferior CIR (HR, 16.64; P < .001) and OS (HR, 4.05; P < .001). NGS-based FLT3-ITD MRD monitoring allows for the identification of patients at high risk for relapse and death following treatment with intensive chemotherapy and midostaurin. Using NGS-based technology, FLT3-ITD emerges as a novel, highly clinically relevant target for MRD monitoring. This trial was registered at www.ClinicalTrials.gov as #NCT01477606.

Targeting USP14/UCHL5: A Breakthrough Approach to Overcoming Treatment-Resistant FLT3-ITD-Positive AML.

FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

Quizartinib: a new hope in acute myeloid leukemia, an applied comprehensive review.

Acute myeloid leukemia (AML) is caused by a defective precursor leading to malignant clonal expansion, often with FMS-like tyrosine kinase-3 receptor (FLT3) mutations, particularly internal tandem duplication (ITD), which has a poor prognosis. Quizartinib, a second-generation FLT3 inhibitor, has FDA approval for relapsed/refractory AML with FLT3/ITD mutation. It has shown promise in clinical studies since 2013 due to its excellent oral absorption and potent activity on FLT3. This review explores Quizartinib's mechanism of action, efficacy in monotherapy or combination with chemotherapy, drug interactions, adverse events, resistance mechanisms and future research directions.

Discovery of SILA-123 as a Highly Potent FLT3 Inhibitor for the Treatment of Acute Myeloid Leukemia with Various FLT3 Mutations.

The FLT3-ITD (internal tandem duplication) mutant has been a promising target for acute myeloid leukemia (AML) drug discovery but is now facing the challenge of resistance due to point mutations. Herein, we have discovered a type II FLT3 inhibitor, SILA-123. This inhibitor has shown highly potent inhibitory effects against FLT3-WT (IC50 = 2.1 nM) and FLT3-ITD (IC50 = 1.0 nM), tumor cells with the FLT3-ITD mutant such as MOLM-13 (IC50 = 0.98 nM) and MV4-11 (IC50 = 0.19 nM), as well as BaF3 cells associated with the FLT3-ITD mutant and point mutations like BaF3-FLT3-ITD-G697R (IC50 = 3.0 nM). Moreover, SILA-123 exhibited promising kinome selectivity against 310 kinases (S score (10) = 0.06). In in vivo studies, SILA-123 significantly suppressed the tumor growth in MV4-11 (50 mg/kg/d, TGI = 87.3%) and BaF3-FLT3-ITD-G697R (50 mg/kg/d, TGI = 60.0%) cell-inoculated allograft models. Our data suggested that SILA-123 might be a promising drug candidate for FLT3-ITD-positive AML.

Distinct FLT3 Pathways Gene Expression Profiles in Pediatric De Novo Acute Lymphoblastic and Myeloid Leukemia with FLT3 Mutations: Implications for Targeted Therapy.

Activating FLT3 mutations plays a crucial role in leukemogenesis, but identifying the optimal candidates for FLT3 inhibitor therapy remains controversial. This study aims to explore the impacts of FLT3 mutations in pediatric acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) and to compare the mutation profiles between the two types to inspire the targeted application of FLT3 inhibitors. We retrospectively analyzed 243 ALL and 62 AML cases, grouping them into FLT3-mutant and wild-type categories, respectively. We then assessed the associations between FLT3 mutations and the clinical manifestations, genetic characteristics, and prognosis in ALL and AML. Additionally, we compared the distinct features of FLT3 mutations between ALL and AML. In ALL patients, those with FLT3 mutations predominantly exhibited hyperdiploidy (48.6% vs. 14.9%, p < 0.001) and higher FLT3 expression (108.02 [85.11, 142.06] FPKM vs. 23.11 [9.16, 59.14] FPKM, p < 0.001), but lower expression of signaling pathway-related genes such as HRAS, PIK3R3, BAD, MAP2K2, MAPK3, and STAT5A compared to FLT3 wild-type patients. There was no significant difference in prognosis between the two groups. In contrast, AML patients with FLT3 mutations were primarily associated with leucocytosis (82.90 [47.05, 189.76] G/L vs. 20.36 [8.90, 55.39] G/L, p = 0.001), NUP98 rearrangements (30% vs. 4.8%, p = 0.018), elevated FLT3 expression (74.77 [54.31, 109.46] FPKM vs. 34.56 [20.98, 48.28] FPKM, p < 0.001), and upregulated signaling pathway genes including PIK3CB, AKT1, MTOR, BRAF, and MAPK1 relative to FLT3 wild-type, correlating with poor prognosis. Notably, internal tandem duplications were the predominant type of FLT3 mutation in AML (66.7%) with higher inserted base counts, whereas they were almost absent in ALL (6.3%, p < 0.001). In summary, our study demonstrated that the forms and impacts of FLT3 mutations in ALL differed significantly from those in AML. The gene expression profiles of FLT3-related pathways may provide a rationale for using FLT3 inhibitors in AML rather than ALL when FLT3 mutations are present.

Lipopolymer/siRNA complexes engineered for optimal molecular and functional response with chemotherapy in FLT3-mutated acute myeloid leukemia

Approximately 25% of newly diagnosed AML patients display an internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene. Although both multi-targeted and FLT3 specific tyrosine kinase inhibitors (TKIs) are being utilized for clinical therapy, drug resistance, short remission periods, and high relapse rates are challenges that still need to be tackled. RNA interference (RNAi), mediated by short interfering RNA (siRNA), presents a mechanistically distinct therapeutic platform with the potential of personalization due to its gene sequence-driven mechanism of action. This study explored the use of a non-viral approach for delivery of FLT3 siRNA (siFLT3) in FLT3-ITD positive AML cell lines and primary cells as well as the feasibility of combining this treatment with drugs currently used in the clinic. Treatment of AML cell lines with FLT3 siRNA nanocomplexes resulted in prominent reduction in cell proliferation rates and induction of apoptosis. Quantitative analysis of relative mRNA transcript levels revealed downregulation of the FLT3 gene, which was accompanied by a similar decline in FLT3 protein levels. Moreover, an impact on leukemic stem cells was observed in a small pool of primary AML samples through significantly reduced colony numbers. An absence of a molecular response post-treatment with lipopolymer/siFLT3 complexes in peripheral blood mononuclear cells, obtained from healthy individuals, denoted a passive selectivity of the complexes towards malignant cells. The effect of combining lipopolymer/siFLT3 complexes with daunorubucin and FLT3 targeting TKI gilteritinib led to a significant augmentation of anti-leukemic activity. These findings demonstrate the promising potential of RNAi implemented with lipopolymer complexes for AML molecular therapy. The study prospectively supports the addition of RNAi therapy to current treatment modalities available to target the heterogeneity prevalent in AML. Statement of significanceWe show that a clinically validated target, the FLT3 gene, can be eradicated in leukemia cells using non-viral RNAi. We validated these lipopolymers as effective vehicles to deliver nucleic acids to leukemic cells. The potency of the lipopolymers was superior to that of the ‘gold-standard’ delivery agent, lipid nanoparticles (LNPs), which are not effective in leukemia cells at clinically relevant doses. Mechanistic studies were undertaken to probe structure-function relationships for effective biomaterial formulations. Cellular and molecular responses to siRNA treatment have been characterized in cell models, including leukemia patient-derived cells. The use of the siRNA therapy with clinically used chemotherapy was demonstrated.

Pediatric Fibromatosis Lacks the Internal Tandem Duplication of EGFR Seen in Congenital Mesoblastic Nephroma.

Classical and mixed congenital mesoblastic nephroma (CMN) are characterized by an internal tandem duplication (ITD) of the EGFR gene, in contrast to cellular CMN that usually harbors an ETV6::NTRK3 gene fusion. This same fusion occurs in infantile fibrosarcoma, and this tumor can be considered as the soft tissue equivalent of cellular CMN. A soft tissue equivalent of classic/mixed CMN remains undefined at the genetic level. Since classical CMN resembles fibromatosis of soft tissue histologically, we asked whether fibromatosis in children might show EGFR ITD. ITD was investigated using the polymerase chain reaction and primers for exons 18 and 25 of the EGFR gene. Seven of the eight cases of classical or mixed CMN were positive by this approach, but none of the five cellular CMNs. Of 11 cases of fibromatosis (six plantar, two digital, and three desmoid), none were positive for EGFR ITD. Within the limits of this small study, we conclude that pediatric fibromatosis is likely not characterized by EGFR ITD. There are isolated reports of pediatric soft tissue tumors that harbor EGFR ITD, but these have the appearance of infantile fibrosarcoma or mixed CMN rather than fibromatosis. We did not find any such cases, since all 14 cases of infantile fibrosarcoma in our study had an ETV6::NTRK3 fusion. The soft tissue tumors with EGFR ITD are not a morphologic match for the low-grade histology of classical CMN. Whether they have a similar favorable biology or behave more like fibrosarcoma with an ETV6::NTRK3 fusion or an alternative fusion involving other kinases remains to be determined.

Advances in the pathogenesis of FLT3 -mutated acute myeloid leukemia and targeted treatments.

FLT3 mutations are among the most common myeloid drivers identified in adult acute myeloid leukemia (AML). Their identification is crucial for the precise risk assessment because of the strong prognostic significance of the most recurrent type of FLT3 alterations, namely internal tandem duplications (ITDs). Recent advances in the pathogenesis and biology of FLT3 -mutated AML have opened an opportunity for development and application of selective inhibition of FLT3 pathway. In the last decade, at least three targeted treatments have been approved by regulatory agencies and several others are currently under investigations. Here, we review the latest advance in the role of FLT3 mutations in AML, providing an outline of the available therapeutic strategies, their mechanisms of actions and of resistance, as well as routes for potential improvement. The availability of FLT3 inhibitors has improved outcomes in AML harboring such mutations, currently also reflected in disease stratification and recommendations. Newer inhibitors are under investigations, and combinations with chemotherapy or other targeted treatments are being explored to further improve disease outcomes.

PTBP1 is a potential indicator of disease progression in acute myeloid leukemia.

This study aimed to verify a novel potential indicator of disease progression in acute myeloid leukemia (AML) patients. Bone marrow samples were collected from 27 AML patients and 27 controls without hematological malignancies. Polypyrimidine tract-binding protein 1 (PTBP1) expression in bone marrow samples was measured, and the association of PTBP1 with the French-American-British (FAB) classification, cytogenetics, risk stratification, and complete remission (CR) rate was analyzed. The correlation between PTBP1 and Ki-67/p53 expression in AML patients was ultimately evaluated. The results showed that PTBP1 mRNA and protein levels were greater in AML patients than in controls. PTBP1 expression was able to distinguish between AML patients and controls (area under the curve, 0.8601; 95% confidence interval, 0.7632-0.9570). Furthermore, PTBP1 expression was associated with an increased frequency of internal tandem duplication mutations within FMS-like tyrosine kinase-3 (FLT3) and a complex karyotype, while PTBP1 expression was not correlated with FAB classification, monosomal karyotype, isolated biallelic CCAAT/enhancer-binding protein α (CEBPA) mutation, or nucleophosmin 1 (NPM1) mutation in patients with AML. Moreover, PTBP1 expression was associated with a poorer prognosis according to risk stratification and a lower CR rate in AML patients. In addition, PTBP1 expression was positively correlated with the expression of the proliferation marker Ki-67 and negatively correlated with the expression of the apoptosis marker p53 in AML patients. Overall, PTBP1 is a viable biomarker that contributes to the risk prediction and the determination of potential drug targets for AML.

Concurrent versus sequential or no triazole anti-fungal therapy in patients undergoing 7 + 3 plus midostaurin induction for FLT-3 acute myelogenous leukemia.

Midostaurin is a multikinase inhibitor approved for the treatment of adult patients with newly diagnosed FMS-like tyrosine kinase 3 mutated (FLT3m) acute myeloid leukemia (AML). Azole antifungal medications are commonly used in AML and are known to interact with anti-cancer drugs such as midostaurin through the CYP3A pathway. However, there are no midostaurin related dose modifications recommended with strong CYP3A inhibitors. We retrospectively reviewed 40 patients between 2017-2022 and compared efficacy and safety outcomes in patients who received azole antifungals concurrently to those who did not receive an azole or received it sequentially to midostaurin for treatment of FLT3m AML. Median age of both groups was approximately 55 years and 70% of patients harbored FLT-3 internal tandem duplication mutations. Most patients in the concurrent arm were on either posaconazole (33%) or isavuconazole (50%) for antifungal prophylaxis and micafungin (72%) for the sequential/no azole arm. Overall CR/CRi rate with concurrent versus sequential/no azole were 72% and 77%, and non-hematologic grade 3 toxicities were 22% and 40% (p = 0.21), respectively. Rates of dose reductions (6% vs. 0%, p = 0.26) and held doses (17% vs. 14%, p = 0.79) were not different between concurrent and sequential/no azole. There were no differences in the rates of new fungal infection during induction between the two groups. Azoles given concurrently or sequentially with midostaurin were found to be equally safe and effective in the treatment of newly diagnosed FLT3 AML. Additional confirmatory studies are needed due to our limited sample size.

Genetic risk stratification and outcomes among treatment-naive patients with AML treated with venetoclax and azacitidine

AbstractThe European LeukemiaNet (ELN) acute myeloid leukemia (AML) genetic risk classification systems are based on response to intensive chemotherapy; their ability to discriminate outcomes in older patients treated with venetoclax-azacitidine may be suboptimal. This pooled analysis of the phase 3 VIALE-A trial (NCT02993523) and phase 1b study (NCT02203773) examined prognostic stratification according to the 2017 and 2022 ELN risk classifications and derived new molecular signatures differentiating venetoclax-azacitidine–treated patients based on overall survival (OS). Overall, 279 patients treated with venetoclax-azacitidine and 113 patients treated with placebo-azacitidine were analyzed. The ELN 2017 or 2022 prognostic criteria classified most patients as adverse-risk AML (60.2% and 72.8% for venetoclax-azacitidine and 65.5% and 75.2% for placebo-azacitidine, respectively). Although outcomes with venetoclax-azacitidine improved across all ELN risk groups compared with placebo-azacitidine, ELN classification systems poorly discriminated venetoclax-azacitidine outcomes. By applying a bioinformatic algorithm, new molecular signatures were derived differentiating OS outcomes with venetoclax-azacitidine. The mutational status of TP53, FLT3 internal tandem duplication (FLT3-ITD), NRAS, and KRAS categorized patients into higher-, intermediate-, and lower-benefit groups (52%, 25%, and 23% of patients, respectively), each associated with a distinct median OS (26.5 months [95% confidence interval (CI), 20.2-32.7]; 12.1 months [95% CI, 7.3-15.2]; and 5.5 months [95% CI, 2.8-7.6], respectively). ELN prognostic classifiers did not provide clinically meaningful risk stratification of OS outcomes in patients treated with venetoclax-azacitidine. TP53, FLT3-ITD, NRAS, and KRAS mutation status allows the classification of these patients into 3 risk groups with distinct differences in median OS. These trials were registered at www.clinicaltrials.gov as #NCT02993523 and #NCT02203773.

Scaffold-hopping of Linifanib to Design 6-phenylisoxazolo[3,4-b]pyridin-3-amine Derivatives as FLT3 Inhibitors for Treating Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the most common type of blood cancer. Fmslike tyrosine kinase 3 (FLT3) is a member of the class III receptor tyrosine kinase family. Overexpression of FLT3 was found in 70-100% of patients with acute myeloid leukaemia. FLT3 internal tandem duplication alteration (ITD) and the tyrosine kinase domain (TKD) are the most common molecular alteration in AML, and FLT3 has become a promising drug target for AML. Objective: A series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives F1–F15 with amide bonds as FLT3 inhibitors were designed and synthesized in order to find a new lead compound to treat AML. Methods: We designed an original scaffold-hopping protocol by combing the RECAP tool with the Gilde-Based Core-Hopping tool to design novel FLT3 inhibitors based on Linifanib. Inhibitors assembled were ranked by the docking scores generated by Glide. Compounds undisclosed among the top 10 were selected to design a series of 6-phenylisoxazolo[3,4-b]pyridin-3-amine derivatives as FLT3 inhibitors. The kinase inhibitory activities of the fifteen compounds were assayed on FLT3 and FLT3-ITD. The antitumor activities of the structurally modified compounds F1–F15 were evaluated against MOLM-13 and MV4-11, typical FLT3-dependent human AML cells carrying FLT3-ITD mutants and the FLT3- independent human cervical carcinoma cell line HL-60 (harboring wide-type FLT3). Results: Structure–activity relationship (SAR) analysis showed that F14 could inhibit FLT3 and FLT3- ITD by 52% and 45.55%, respectively, at the concentration of 1 mΜ. F14 exhibited potent activity against FLT3-dependent human acute myeloid leukemia (AML) cell lines, MOLM-13, and MV4-11 (harboring FLT3-ITD mutant) with IC50 values of 2.558 μM and 1.785 μM, respectively. Conclusion: F14 could be used as a novel lead compound to further develop FLT3 inhibitors against AML with FLT3-ITD mutant.