KIT and FLT3-ITD Mutations Do Not Predict Outcomes in Pediatric Core-Binding Factor Acute Myeloid Leukemia: Findings from the C-HUANAN-AML-15 Multicenter Cohort Study

KIT and FLT3-ITD Mutations Do Not Predict Outcomes in Pediatric Core-Binding Factor Acute Myeloid Leukemia: Findings from the C-HUANAN-AML-15 Multicenter Cohort Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article KIT and FLT3-ITD Mutations Do Not Predict Outcomes in Pediatric Core-Binding Factor Acute Myeloid Leukemia: Findings from the C-HUANAN-AML-15 Multicenter Cohort Study Yongzhi Zheng, Lihua Yu, Shaohua Le, Nainong Li, Xiaoqin Feng, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8473547/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background Although core-binding factor acute myeloid leukemia (CBF-AML) is generally considered a favorable-risk subtype in children, disease relapse remains a significant concern. The prognostic relevance of co-occurring mutations, particularly KIT and FLT3-ITD , remains debatable, and treatment intensity may modulate their impact. Methods This multicenter analysis included 289 children (< 14 years) with newly diagnosed CBF-AML enrolled in the C-HUANAN-AML-15 study (2015–2023). KIT and FLT3-ITD mutations were identified via cytogenetic analysis and targeted sequencing. Measurable residual disease (MRD) was evaluated by multiparameter flow cytometry (MFC) and quantitative polymerase chain reaction (PCR) following induction chemotherapy. Survival analyses were performed using Kaplan–Meier and Cox regression methods. Results KIT mutations were detected in 103 patients (35.6%), predominantly involving exon 17 (69.9%), and were associated with extramedullary disease, sex chromosome loss, and trisomy 22. No significant differences in 5-year event-free survival (EFS), overall survival (OS), or cumulative incidence of relapse (CIR) were observed between patients with and without KIT mutations. FLT3 -ITD mutations (5.5% of patients) did not adversely affect outcomes. Neither mutation independently predicted survival. MRD positivity (MFC-MRD ≥ 0.1%) after the second induction cycle strongly predicted inferior EFS and OS and higher CIR, with corresponding results observed for molecular MRD and parallel findings for PCR-based MRD. Conclusions In this large multicenter cohort, KIT and FLT3 -ITD mutations did not adversely affect the prognosis of pediatric CBF-AML treated according to the C-HUANAN-AML-15 protocol. MRD after induction was the most powerful predictor of relapse and survival, underscoring its importance for risk stratification in future pediatric AML trials. Pediatric acute myeloid leukemia Core-binding factor acute myeloid leukemia KIT mutations FLT3 internal tandem duplication Measurable residual disease Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Core-binding factor acute myeloid leukemia (CBF-AML) represents a distinct subtype of AML defined by recurrent cytogenetic rearrangements, most commonly RUNX1::RUNX1T1 and CBFβ::MYH11 . It accounts for approximately 25%–37% of pediatric AML cases, making it the predominant cytogenetic subgroup [ 1 – 3 ]. Although t(8;21) and inv(16) are linked to relatively favorable outcomes and categorized as low-risk [ 4 – 6 ], 20% to 40% of children with CBF-AML ultimately relapse, underscoring the need to identify potential molecular determinants of relapse, including co-occurring variants [ 7 – 10 ]. Somatic mutations co-occurring with these fusion genes act as drivers of leukemogenesis and may modify outcomes [ 1 , 11 ]. Among these, activating mutations in receptor tyrosine kinases (RTKs), particularly in KIT and FLT3 , are frequently observed. Several studies have indicated that specific KIT mutations, especially those in exon 17, and the presence of FLT3-ITD are associated with elevated relapse rates and inferior survival in both adult and pediatric CBF-AML [ 1 , 9 , 10 , 12 – 19 ], whereas other studies have not demonstrated an adverse prognostic effect [ 20 – 23 ]. These discrepancies may reflect differences in cohort size, mutation type, and treatment intensity. Notably, emerging evidence from adult studies suggests that intensive chemotherapy regimens, particularly those incorporating high-dose cytarabine and FLAG in combination with either gemtuzumab ozogamicin (GO) or idarubicin (IDA), may mitigate the historically adverse prognosis associated with RTK mutations [ 21 , 24 , 25 ]. The FLAG-IDA regimen has demonstrated superior complete remission rates and survival outcomes compared with standard protocols in newly diagnosed adult AML [ 25 – 27 ]. However, its use as frontline induction therapy in pediatric AML remains limited [ 28 , 29 ]. The C-HUANAN-AML-15 protocol, which employs FLAG-IDA induction, provides a contemporary cohort to evaluate the prognostic significance of KIT and FLT3-ITD mutations within the context of modern, intensified therapy. This study aimed to assess the impact of these genetic lesions on outcomes in a large, uniformly treated pediatric CBF-AML cohort. Methods Patients This multicenter, retrospective analysis enrolled pediatric patients aged < 14 years with newly diagnosed CBF-AML between January 2015 and December 2023. Participants were treated uniformly according to the C-HUANAN-AML-15 protocol across 11 tertiary medical centers in Southern China. Diagnosis was based on morphology and flow cytometry, following World Health Organization (WHO) criteria [ 30 ]. Patients with Down syndrome or a history of prior cytotoxic chemotherapy were excluded. The study received ethical approval from the relevant Institutional Review Board, with informed consent waived in compliance with national regulations and the Declaration of Helsinki. C-HUANAN AML 2015 Protocol The C-HUANAN-AML-15 protocol [ 31 ], adapted from the UK MRC AML15 framework [ 26 ], comprised four sequential cycles: two induction blocks, followed by two consolidation phases. Induction therapy consisted of either FLAG-IDA (fludarabine, cytarabine, G-CSF, and idarubicin) or DAE (daunorubicin, cytarabine, etoposide), assigned non-randomly based on physician discretion and institutional guidelines. Subsequent consolidation included homoharringtonine combined with cytarabine, and mitoxantrone with cytarabine. The treatment schematics are shown in Online Resource 1 . Cytogenetic and Molecular Analyses Bone marrow (BM) cells underwent short-term culture (24–48 h) without stimulation, with karyotyping performed on ≥ 20 metaphase cells via G-banding. RUNX1::RUNX1T1 and CBFB::MYH11 transcript levels were measured using TaqMan-based real-time quantitative reverse transcriptase polymerase chain reaction (PCR) and expressed as the ratio of target transcript copies to ABL1 transcript copies in BM samples [ 18 ]. KIT mutations (exons 8 and 17) were screened by PCR until December 2018 [ 19 ], and then by whole exome sequencing (WES) on an Illumina platform (Illumina, USA), as previously described [ 32 ]. FLT3 -ITD detection involved PCR of exons 14–15, followed by capillary electrophoresis on an ABI 3130 (Applied Biosystems) and analysis via GeneMapper; positivity was defined as an abnormal peak exceeding the wild-type signal. Measurable Residual Disease (MRD) MRD was assessed at two predefined time points: 28–35 days post-first induction (time point 1, TP1) and pre-consolidation post-second induction (time point 2, TP2). MRD was evaluated using multiparameter flow cytometry (MFC) and quantitative PCR (qPCR), with a threshold of < 0.1% considered negative. Definitions Complete remission (CR) was defined as < 5% marrow blasts with hematologic recovery. The events included relapse, death, therapy abandonment, or secondary malignancy. Overall survival (OS) was measured from diagnosis to death or last follow-up. Event-free survival (EFS) was calculated from diagnosis to the first event or last follow-up. The cumulative incidence of relapse (CIR) was defined as the time from CR to relapse, with death in the absence of relapse considered a competing event. Patients were followed up until death, the last contact, or censoring at the study cutoff (April 1, 2024). Statistical Analysis Data were analyzed using SPSS 28.0, and graphical representations were generated using GraphPad Prism. Continuous variables are presented as median (range) and were compared using the Mann–Whitney U-test. Categorical variables were analyzed using the χ ² test or Fisher’s exact test, as appropriate. Survival endpoints (OS, EFS) were estimated via the Kaplan–Meier method with log-rank comparisons. CIR was evaluated using competing risks analysis (Gray’s test). Prognostic factors were identified through univariate Cox regression (variables with P < 0.05), followed by multivariable Cox proportional hazards modeling. All statistical tests were bilateral, with a significance threshold of P < 0.05. Results Clinical and Genetic Characteristics of the Pediatric CBF-AML Cohort​​ Among a total of 875 pediatric patients with de novo AML (excluding acute promyelocytic leukemia), 289 (33.0%) were diagnosed with CBF-AML. Within this subgroup, 231 (79.9%) harbored the RUNX1::RUNX1T1 fusion, while 58 (20.1%) carried CBFβ::MYH11 . Compared with patients with non-CBF-AML, those with CBF-AML demonstrated a significantly higher prevalence of age ≥ 10 years, extramedullary involvement, and KIT mutations, whereas initial white blood cell counts ≥ 50×10⁹/L and FLT3 -ITD mutations were less frequently observed (all P < 0.05). Detailed baseline characteristics are summarized in Online Resource 2 . The CBF-AML cohort also demonstrated significantly higher CR rates after induction, superior 5-year OS and EFS, and a significantly lower 5-year CIR compared with the non-CBF-AML group ( Online Resource 3 ). Spectrum and Distribution of KIT Mutations​​ KIT mutations were detected in 103 (35.6%) of 289 patients with CBF-AML, with comparable frequencies between RUNX1::RUNX1T1 (35.5%) and CBFβ::MYH11 (36.2%) subgroups. A total of 117 mutations were identified, with exon 17 alterations being most prevalent (69.9%), followed by exon 8 mutations (29.1%) ( Online Resource 4 ). Exon 8 mutations occurred more frequently in CBFβ::MYH11 AML (9/22, 40.9%) than in RUNX1::RUNX1T1 AML (13/59, 22.1%), whereas exon 17 mutations predominated in RUNX1::RUNX1T1 AML (66/95, 69.5%) compared with CBFβ::MYH11 AML (13/22, 59.1%), although neither difference reached statistical significance ( P = 0.069 and P = 0.349, respectively). At the residue level, D816 and N822 were the most commonly affected in exon 17, occurring in 36 (50.0%) and 32 (44.4%) patients, respectively. By contrast, exon 8 mutations at T417 were significantly enriched in CBFβ::MYH11 AML (5/22, 22.7%) compared with RUNX1::RUNX1T1 AML (5/95, 5.3%; P = 0.020), whereas N822K mutations in exon 17 were more frequent in RUNX1::RUNX1T1 AML (31/95, 32.6%) than in CBFβ::MYH11 AML (2/22, 9.1%; P = 0.034). Compared with patients without KIT mutations, those with KIT mutations showed significantly higher frequencies of extramedullary involvement, loss of the X/Y chromosome, and trisomy 22 ( Online Resource 5 ). Prognostic Impact of KIT Mutations in Pediatric Patients with CBF-AML At a median follow-up of 44.7 months (range, 0.8–109.9), the 5-year EFS, OS, and CIR among pediatric patients with KIT mutations were 85.6% ± 3.6%, 91.1% ± 3.0%, and 7.0% ± 2.8%, respectively. These outcomes did not significantly differ from those of KIT wild-type patients (EFS, 77.9% ± 3.1%, P = 0.177; OS, 85.5% ± 2.8%, P = 0.377; CIR, 11.6% ± 2.6%, P = 0.303) (Fig. 1 ). Subgroup analyses further confirmed that the presence or location of KIT mutations did not significantly influence prognosis: patients with exon 17 mutations had comparable 5-year EFS, OS, and CIR to those with other KIT mutations or wild-type KIT (EFS, 85.3% ± 4.3% vs. 86.4% ± 6.3% vs. 77.9% ± 3.1%, P = 0.395; OS, 88.7% ± 4.1% vs. 96.4% ± 3.6% vs. 85.5% ± 2.8%, P = 0.298; CIR, 5.1% ± 2.9% vs. 10.8%± 5.9% vs. 11.6% ± 2.6%, P = 0.425) (Fig. 2 ). Similarly, no significant survival differences were observed between KIT -mutated and wild-type patients within either the RUNX1::RUNX1T1 or CBFβ::MYH11 fusion subgroups ( Online Resources 6 and 7 ). Among pediatric patients with KIT mutations, outcomes were also comparable between fusion subtypes (EFS, 87.0% ± 3.9% vs. 80.4% ± 8.8%, P = 0.313; OS, 90.5% ± 6.4% vs. 91.4% ± 3.4%, P = 0.326; CIR, 7.1% ± 3.1% vs. 6.7% ± 6.4%, P = 0.936) ( Online Resource 8 ), and between those receiving FLAG-IDA versus DAE induction regimens (EFS, 85.9% ± 4.0% vs. 84.2% ± 8.4%, P = 0.773; OS, 91.6% ± 3.3% vs. 88.9% ± 7.4%, P = 0.706; CIR, 8.4% ± 3.3% vs. 0%, P = 0.255) ( Online Resource 9 ). Prevalence and Prognostic Impact of FLT3-ITD Mutations​​ FLT3 -ITD mutations were identified in 16 (5.5%) pediatric patients with CBF-AML, with a higher incidence among those carrying RUNX1::RUNX1T1 (6.1%, 14/231) than in CBFβ::MYH11 cases (3.4%, 2/58). No significant differences in baseline characteristics were observed between FLT3- ITD–positive and –negative patients ( Online Resource 10). Among the 16 CBF-AML patients harboring FLT3 -ITD, two discontinued treatment after the first induction course, and one died from infectious complications following the second induction. Of the 13 evaluable patients, four underwent hematopoietic stem cell transplantation (HSCT) during their first complete remission (CR1), and all remained event-free. The remaining nine patients received consolidation chemotherapy alone, with only one relapse and eight event-free survivors. Outcomes in FLT3 -ITD–positive patients were not inferior to those without this mutation, with comparable 5-year EFS (75.0% ± 10.8% vs. 80.8% ± 2.5%, p = 0.438), OS (87.1% ± 8.6% vs. 86.4% ± 2.4%, p = 0.821), and CIR (8.4% ± 3.3% vs. 10.1% ± 2.0%, p = 0.800) (Fig. 3 ). Although not statistically significant, patients with CBF-AML and FLT3 -ITD showed a trend toward better survival than those with non-CBF-AML harboring FLT3 -ITD ( Online Resource 11 ). After excluding patients who underwent HSCT in CR1, CBF-AML patients with FLT3- ITD demonstrated significantly superior 5-year EFS (75.0% ± 10.8% vs. 17.8% ± 6.5%; P = 0.003) and OS (82.5% ± 11.3% vs. 46.0% ± 8.9%; P = 0.049), alongside a lower CIR (75.0% ± 12.5% vs. 70.6% ± 9.9%; P = 0.005), compared to their non-CBF-AML counterparts (Fig. 4 ). Risk-Factor Analysis in Pediatric Patients with CBF-AML Ten patients (3.5%) were excluded due to treatment discontinuation or transfer between institutions before the second induction, leaving 279 patients evaluable for the intention-to-treat (ITT) analysis. Univariate Cox regression was performed to evaluate the impact of baseline clinical and genetic variables, including age, sex, initial white blood cell (WBC) count, extramedullary involvement, fusion type (RUNX1::RUNX1T1 or CBFβ::MYH11 ), additional cytogenetic abnormalities (− X/−Y), and induction regimen (FLAG-IDA vs. DAE), on long-term survival. None of these factors were significantly associated with long-term outcomes ( Online Resource 12 ). In contrast, MRD status at TP2 emerged as a powerful prognostic indicator. Among 268 patients with available MRD data at TP2, those exhibiting MFC-MRD ≥ 0.1% experienced significantly inferior 5-year EFS (70.1% ± 7.3% vs. 87.3% ± 2.3%, P = 0.001) and OS (74.2% ± 7.1% vs. 92.1% ± 2.0%, P < 0.001), and higher 5-year CIR (25.9% ± 7.1% vs. 7.3% ± 1.9%, P < 0.001), compared with those who were MRD-negative (Fig. 5 ). Similarly, molecular MRD ≥ 0.1% was associated with worse EFS (75.6% ± 4.6% vs. 89.8% ± 2.4%, P = 0.003), OS (80.8% ± 4.4% vs. 92.9% ± 2.2%, P = 0.003), and higher CIR (20.1% ± 4.4% vs. 4.7% ± 1.7%, P < 0.001) (Fig. 6 ). Discussion This large multicenter analysis of pediatric patients with CBF-AML uniformly treated according to the C-HUANAN-AML-15 protocol revealed that the prognostic significance of receptor tyrosine kinase (RTK) mutations, specifically in KIT and FLT3-ITD , is substantially attenuated in the context of intensive chemotherapy. In contrast, MRD after induction emerged as the dominant predictor of clinical outcomes, underscoring the value of dynamic risk assessment over static genetic profiling in this population. Consistent with prior reports, KIT mutations were present in approximately one-third of pediatric patients with CBF-AML, with exon 17 alterations being most prevalent [ 2 , 9 , 19 , 20 , 33 , 34 ]. The prognostic implications of KIT mutations in pediatric CBF-AML remain debated. Historically, KIT exon 17 mutations—particularly D816 and N822—have been associated with higher relapse risk [ 2 , 10 , 17 , 19 , 35 ]. However, conflicting evidence exists, with several reports failing to confirm an independent adverse prognostic impact [ 16 , 20 – 23 ]. Functional analyses further suggest that the clinical behavior of KIT mutations may depend on the mutation class (e.g., gain-of-function vs. kinase domain alterations) [ 14 ]. In the present study, KIT mutations, regardless of subtype or fusion partner, did not significantly affect EFS, OS, or CIR. This lack of prognostic influence may stem from intensified frontline therapy, predominantly FLAG-IDA, which provides superior antileukemic activity compared to conventional “7 + 3” regimens. Supporting this, adult CBF-AML studies have shown that fludarabine-containing protocols, such as FLAG-IDA or FLAG combined with gemtuzumab ozogamicin (GO), can mitigate the negative effects of KIT mutations [ 21 , 23 , 26 ]. Our results align with this, suggesting that uniform FLAG-IDA treatment likely neutralizes KIT -driven leukemogenesis. Discrepancies across studies may be attributable to variations in sample size, consolidation regimens, and MRD integration, as noted in recent reviews [ 2 ]. FLT3 -ITD mutations were infrequent in our cohort (5.5%), consistent with their lower prevalence in CBF-AML than in other AML subtypes [ 11 , 22 ]. The prognostic relevance of FLT3 -ITD in CBF-AML appears to differ between adults and children. In adults, FLT3 -ITD confers inferior outcomes and challenges favorable-risk classification, though HSCT in CR1 yields limited benefit [ 36 , 37 ]. In contrast, pediatric studies indicate minimal impact. For example, the Japanese Childhood AML Cooperative Study Group found that KIT —but not FLT3 -ITD—was strongly associated with relapse in t(8;21) cases [ 15 ], a finding echoed in European and US cooperative series [ 11 , 20 ]. In the present study, FLT3 -ITD did not confer an adverse impact on survival in pediatric CBF-AML. Interestingly, the outcomes for CBF-AML patients harboring FLT3 -ITD were at least comparable and possibly superior to those of non-CBF-AML patients with FLT3 -ITD. When patients undergoing HSCT in CR1 were excluded, CBF-AML patients with FLT3 -ITD showed significantly higher EFS and OS and lower CIR than their non-CBF-AML counterparts, suggesting that CBF cytogenetics may buffer the oncogenic effects of FLT3 -ITD. Among 13 evaluable patients with FLT3 -ITD, all four receiving CR1-HSCT remained event-free, while only one of nine on chemotherapy alone relapsed, suggesting that chemotherapy may be sufficient in this context and routine upfront HSCT may not be necessary. However, the small sample size (n = 16) limits the statistical power; thus, larger prospective MRD-integrated studies are needed to confirm whether pediatric CBF-AML with FLT3 -ITD can safely avoid transplantation. In pediatric AML, MRD monitoring has revolutionized risk stratification, with post-induction levels guiding therapy intensification and reducing overtreatment [ 4 , 38 ]. Across both fusion subtypes, MRD after induction is the most robust predictor of outcome. Patients with MFC- or PCR-based MRD ≥ 0.1% had significantly poor survival, consistent with multiple pediatric AML studies [ 39 , 40 ]. These findings reinforce that MRD is a stronger prognostic marker than baseline mutations and should guide post-remission therapy. This study has several limitations. First, despite being one of the largest multicenter pediatric CBF-AML cohorts in China, the sample size of patients harboring FLT3-ITD mutations was small (n = 16), limiting the statistical power to draw definitive conclusions regarding their prognostic significance. Second, the non-randomized assignment of patients to FLAG-IDA versus DAE induction regimens introduces potential selection bias. Third, although MRD was systematically assessed, inter-laboratory variability across participating centers may have affected the uniformity of detection thresholds. These limitations highlight the need for larger, prospective, and ideally international collaborative studies integrating genomic and MRD profiling to refine risk stratification and therapeutic strategies for pediatric CBF-AML. In conclusion, our findings suggest that within the framework of contemporary intensive chemotherapy, the historically adverse prognosis associated with KIT and FLT3 -ITD mutations in pediatric CBF-AML may be neutralized. MRD levels after induction remain the most powerful prognostic biomarker, emphasizing the need for dynamic response-based risk stratification in future clinical trials. Declarations Acknowledgements We express our deepest appreciation to the patients who donated samples. We also extend our sincere gratitude to the physicians from the participating institutions for providing clinical data. Chunfu Li served as the principal investigator of the C-HUANAN-AML-15 protocol, and Xiaoqun Feng was the chairperson of the C-HUANAN-AML working party. Competing Interests There are no conflicts of interest to declare. Funding This study was supported by the Fujian Provincial Clinical Research Center for Hematological Malignancies (Grant No. 2020Y2006) and the Natural Science Foundation of Fujian Province (Grant No. 2025J01122). Ethics Approval and Consent to Participate Ethical Approval No obtained from the Fujian Medical University Union Hospital Institutional Review Board (Approval No.: 2025KY666), with a waiver of informed consent granted in accordance with the national regulations and the Declaration of Helsinki. Consent for Publication Not applicable. Data Availability The data supporting the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Author Contributions Conceptualization: Y.Z., L.Yu, and L.Yang Data curation: S.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C., and L.Yang Formal analysis: Y.Z. and L.Yu Funding acquisition: N.L. and Y.Z. Investigation: Y.Z., L.Yu, and L.Yang Methodology: Y.Z., L.Yu, and L.Yang Project administration: Y.Z., L.Yu, and L.Yang Resources: S.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C., and L.Yang Software: Y.Z. and L.Yu Supervision: L.Yang Validation: All authors Visualization: Y.Z. and L.Yu Writing – original draft: Y.Z. and L.Yu Writing – review & editing: S.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C., L.Yang References Duployez N, Marceau-Renaut A, Boissel N et al (2016) Comprehensive mutational profiling of core binding factor acute myeloid leukemia. Blood 127(20):2451–2459. https://doi.org/10.1182/blood-2015-12-688705 Hu Z, Tang X, Chen F et al (2025) Molecular genetics profiling of core-binding factor acute myeloid leukemia in pediatrics. 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Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 10 May, 2026 Reviews received at journal 10 Apr, 2026 Reviews received at journal 06 Apr, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers invited by journal 06 Jan, 2026 Editor assigned by journal 02 Jan, 2026 Submission checks completed at journal 02 Jan, 2026 First submitted to journal 29 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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05:50:45","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":128031,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/00eb1276afccda4c1145ee2f.html"},{"id":100361808,"identity":"9b18b8bc-4e56-43a1-978c-6f063aa6bd8b","added_by":"auto","created_at":"2026-01-16 07:45:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":170773,"visible":true,"origin":"","legend":"\u003cp\u003ePrognostic impact of \u003cem\u003eKIT \u003c/em\u003emutations in pediatric patients with core-binding factor acute myeloid leukemia (CBF-AML). Kaplan–Meier curves for (a) overall survival (OS), (b) event-free survival (EFS), and (c) cumulative incidence of relapse (CIR)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/865fceb7bc699bf2fd82d620.png"},{"id":100360897,"identity":"9ef32ada-a578-4fb6-bcbd-937222f6f382","added_by":"auto","created_at":"2026-01-16 07:44:09","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":199996,"visible":true,"origin":"","legend":"\u003cp\u003eComparative outcomes of \u003cem\u003eKIT\u003c/em\u003e exon 17 mutations versus other \u003cem\u003eKIT\u003c/em\u003e exon mutations and KIT wild-type in pediatric patients with CBF-AML. Kaplan–Meier curves for (a) OS and (b) EFS, and competing risks analysis for (c) CIR\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/08737279b75509e19d0b47dc.png"},{"id":100007649,"identity":"0637256b-ae2d-4e03-b7cd-c7793ecb907b","added_by":"auto","created_at":"2026-01-12 05:50:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163175,"visible":true,"origin":"","legend":"\u003cp\u003ePrognostic impact of \u003cem\u003eFLT3-\u003c/em\u003eITD\u003cem\u003e \u003c/em\u003emutations in pediatric patients with CBF-AML. Kaplan–Meier curves for (a) OS, (b) EFS, and (c) CIR\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/40fce030356d89557fd0ba26.png"},{"id":100007660,"identity":"384c2ba2-caa2-4275-9f46-b745efcac962","added_by":"auto","created_at":"2026-01-12 05:50:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":188187,"visible":true,"origin":"","legend":"\u003cp\u003eComparative outcomes of patients with CBF-AML harboring \u003cem\u003eFLT3\u003c/em\u003e-ITD versus those with non-CBF-AML and \u003cem\u003eFLT3\u003c/em\u003e-ITD, excluding patients who underwent hematopoietic stem cell transplantation in first complete remission. Kaplan–Meier estimates for (a) EFS, (b) OS, and (c) CIR\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/ac6afe0b64cd002429bcc5ff.png"},{"id":100007653,"identity":"c3f66f18-2409-4f44-beff-75dcd5381647","added_by":"auto","created_at":"2026-01-12 05:50:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":185482,"visible":true,"origin":"","legend":"\u003cp\u003ePrognostic impact of measurable residual disease (MRD) evaluated by multiparameter flow cytometry (MFC-MRD) after the second induction course (time point 2 [TP2]) in pediatric patients with CBF-AML. Kaplan–Meier curves for (a) OS, (b) EFS, and (c) CIR\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/9d950a0e43938385004d7dc2.png"},{"id":100360854,"identity":"6d404524-eb79-4d54-883d-f53f586943fe","added_by":"auto","created_at":"2026-01-16 07:44:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":168855,"visible":true,"origin":"","legend":"\u003cp\u003ePrognostic impact of molecular MRD evaluated by quantitative PCR (qPCR) after the second induction course (time point 2 [TP2]) in pediatric patients with CBF-AML. Kaplan–Meier curves for (a) OS, (b) EFS, and (c) CIR\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/778b210b2e20a073b00ebe7e.png"},{"id":100380764,"identity":"6e8038cc-d1d9-48c6-8e41-7d972fc400eb","added_by":"auto","created_at":"2026-01-16 10:33:13","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1998187,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/2f913f4d-a567-4c3b-a434-cf5fb4d35194.pdf"},{"id":100007651,"identity":"4d5dc201-cee0-4946-bd02-ea8508eb697f","added_by":"auto","created_at":"2026-01-12 05:50:44","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":503552,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8473547/v1/7d00e3c0468494f2bc36791a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"KIT and FLT3-ITD Mutations Do Not Predict Outcomes in Pediatric Core-Binding Factor Acute Myeloid Leukemia: Findings from the C-HUANAN-AML-15 Multicenter Cohort Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCore-binding factor acute myeloid leukemia (CBF-AML) represents a distinct subtype of AML defined by recurrent cytogenetic rearrangements, most commonly \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e and \u003cem\u003eCBFβ::MYH11\u003c/em\u003e. It accounts for approximately 25%\u0026ndash;37% of pediatric AML cases, making it the predominant cytogenetic subgroup [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Although t(8;21) and inv(16) are linked to relatively favorable outcomes and categorized as low-risk [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], 20% to 40% of children with CBF-AML ultimately relapse, underscoring the need to identify potential molecular determinants of relapse, including co-occurring variants [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSomatic mutations co-occurring with these fusion genes act as drivers of leukemogenesis and may modify outcomes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Among these, activating mutations in receptor tyrosine kinases (RTKs), particularly in \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3\u003c/em\u003e, are frequently observed. Several studies have indicated that specific \u003cem\u003eKIT\u003c/em\u003e mutations, especially those in exon 17, and the presence of \u003cem\u003eFLT3-ITD\u003c/em\u003e are associated with elevated relapse rates and inferior survival in both adult and pediatric CBF-AML [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], whereas other studies have not demonstrated an adverse prognostic effect [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These discrepancies may reflect differences in cohort size, mutation type, and treatment intensity.\u003c/p\u003e \u003cp\u003eNotably, emerging evidence from adult studies suggests that intensive chemotherapy regimens, particularly those incorporating high-dose cytarabine and FLAG in combination with either gemtuzumab ozogamicin (GO) or idarubicin (IDA), may mitigate the historically adverse prognosis associated with RTK mutations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The FLAG-IDA regimen has demonstrated superior complete remission rates and survival outcomes compared with standard protocols in newly diagnosed adult AML [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, its use as frontline induction therapy in pediatric AML remains limited [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The C-HUANAN-AML-15 protocol, which employs FLAG-IDA induction, provides a contemporary cohort to evaluate the prognostic significance of \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3-ITD\u003c/em\u003e mutations within the context of modern, intensified therapy. This study aimed to assess the impact of these genetic lesions on outcomes in a large, uniformly treated pediatric CBF-AML cohort.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003eThis multicenter, retrospective analysis enrolled pediatric patients aged\u0026thinsp;\u0026lt;\u0026thinsp;14 years with newly diagnosed CBF-AML between January 2015 and December 2023. Participants were treated uniformly according to the C-HUANAN-AML-15 protocol across 11 tertiary medical centers in Southern China. Diagnosis was based on morphology and flow cytometry, following World Health Organization (WHO) criteria [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Patients with Down syndrome or a history of prior cytotoxic chemotherapy were excluded. The study received ethical approval from the relevant Institutional Review Board, with informed consent waived in compliance with national regulations and the Declaration of Helsinki.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eC-HUANAN AML 2015 Protocol\u003c/h3\u003e\n\u003cp\u003eThe C-HUANAN-AML-15 protocol [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], adapted from the UK MRC AML15 framework [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], comprised four sequential cycles: two induction blocks, followed by two consolidation phases. Induction therapy consisted of either FLAG-IDA (fludarabine, cytarabine, G-CSF, and idarubicin) or DAE (daunorubicin, cytarabine, etoposide), assigned non-randomly based on physician discretion and institutional guidelines. Subsequent consolidation included homoharringtonine combined with cytarabine, and mitoxantrone with cytarabine. The treatment schematics are shown in \u003cb\u003eOnline Resource 1\u003c/b\u003e.\u003c/p\u003e\n\u003ch3\u003eCytogenetic and Molecular Analyses\u003c/h3\u003e\n\u003cp\u003eBone marrow (BM) cells underwent short-term culture (24\u0026ndash;48 h) without stimulation, with karyotyping performed on \u0026ge;\u0026thinsp;20 metaphase cells via G-banding. \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e and \u003cem\u003eCBFB::MYH11\u003c/em\u003e transcript levels were measured using TaqMan-based real-time quantitative reverse transcriptase polymerase chain reaction (PCR) and expressed as the ratio of target transcript copies to \u003cem\u003eABL1\u003c/em\u003e transcript copies in BM samples [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eKIT\u003c/em\u003e mutations (exons 8 and 17) were screened by PCR until December 2018 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and then by whole exome sequencing (WES) on an Illumina platform (Illumina, USA), as previously described [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eFLT3\u003c/em\u003e-ITD detection involved PCR of exons 14\u0026ndash;15, followed by capillary electrophoresis on an ABI 3130 (Applied Biosystems) and analysis via GeneMapper; positivity was defined as an abnormal peak exceeding the wild-type signal.\u003c/p\u003e\n\u003ch3\u003eMeasurable Residual Disease (MRD)\u003c/h3\u003e\n\u003cp\u003eMRD was assessed at two predefined time points: 28\u0026ndash;35 days post-first induction (time point 1, TP1) and pre-consolidation post-second induction (time point 2, TP2). MRD was evaluated using multiparameter flow cytometry (MFC) and quantitative PCR (qPCR), with a threshold of \u0026lt;\u0026thinsp;0.1% considered negative.\u003c/p\u003e\n\u003ch3\u003eDefinitions\u003c/h3\u003e\n\u003cp\u003eComplete remission (CR) was defined as \u0026lt;\u0026thinsp;5% marrow blasts with hematologic recovery. The events included relapse, death, therapy abandonment, or secondary malignancy. Overall survival (OS) was measured from diagnosis to death or last follow-up. Event-free survival (EFS) was calculated from diagnosis to the first event or last follow-up. The cumulative incidence of relapse (CIR) was defined as the time from CR to relapse, with death in the absence of relapse considered a competing event. Patients were followed up until death, the last contact, or censoring at the study cutoff (April 1, 2024).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using SPSS 28.0, and graphical representations were generated using GraphPad Prism. Continuous variables are presented as median (range) and were compared using the Mann\u0026ndash;Whitney U-test. Categorical variables were analyzed using the χ\u003csup\u003e\u0026sup2;\u003c/sup\u003e test or Fisher\u0026rsquo;s exact test, as appropriate. Survival endpoints (OS, EFS) were estimated via the Kaplan\u0026ndash;Meier method with log-rank comparisons. CIR was evaluated using competing risks analysis (Gray\u0026rsquo;s test). Prognostic factors were identified through univariate Cox regression (variables with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), followed by multivariable Cox proportional hazards modeling. All statistical tests were bilateral, with a significance threshold of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eClinical and Genetic Characteristics of the Pediatric CBF-AML Cohort​​\u003c/h2\u003e \u003cp\u003eAmong a total of 875 pediatric patients with de novo AML (excluding acute promyelocytic leukemia), 289 (33.0%) were diagnosed with CBF-AML. Within this subgroup, 231 (79.9%) harbored the \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e fusion, while 58 (20.1%) carried \u003cem\u003eCBFβ::MYH11\u003c/em\u003e. Compared with patients with non-CBF-AML, those with CBF-AML demonstrated a significantly higher prevalence of age\u0026thinsp;\u0026ge;\u0026thinsp;10 years, extramedullary involvement, and \u003cem\u003eKIT\u003c/em\u003e mutations, whereas initial white blood cell counts\u0026thinsp;\u0026ge;\u0026thinsp;50\u0026times;10⁹/L and \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations were less frequently observed (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Detailed baseline characteristics are summarized in \u003cb\u003eOnline Resource 2\u003c/b\u003e. The CBF-AML cohort also demonstrated significantly higher CR rates after induction, superior 5-year OS and EFS, and a significantly lower 5-year CIR compared with the non-CBF-AML group (\u003cb\u003eOnline Resource 3\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eSpectrum and Distribution of KIT Mutations​​\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKIT\u003c/em\u003e mutations were detected in 103 (35.6%) of 289 patients with CBF-AML, with comparable frequencies between \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e (35.5%) and \u003cem\u003eCBFβ::MYH11\u003c/em\u003e (36.2%) subgroups. A total of 117 mutations were identified, with exon 17 alterations being most prevalent (69.9%), followed by exon 8 mutations (29.1%) (\u003cb\u003eOnline Resource 4\u003c/b\u003e). Exon 8 mutations occurred more frequently in \u003cem\u003eCBFβ::MYH11\u003c/em\u003e AML (9/22, 40.9%) than in \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e AML (13/59, 22.1%), whereas exon 17 mutations predominated in \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e AML (66/95, 69.5%) compared with \u003cem\u003eCBFβ::MYH11\u003c/em\u003e AML (13/22, 59.1%), although neither difference reached statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.069 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.349, respectively). At the residue level, D816 and N822 were the most commonly affected in exon 17, occurring in 36 (50.0%) and 32 (44.4%) patients, respectively. By contrast, exon 8 mutations at T417 were significantly enriched in \u003cem\u003eCBFβ::MYH11\u003c/em\u003e AML (5/22, 22.7%) compared with \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e AML (5/95, 5.3%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020), whereas N822K mutations in exon 17 were more frequent in \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e AML (31/95, 32.6%) than in \u003cem\u003eCBFβ::MYH11\u003c/em\u003e AML (2/22, 9.1%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.034). Compared with patients without \u003cem\u003eKIT\u003c/em\u003e mutations, those with \u003cem\u003eKIT\u003c/em\u003e mutations showed significantly higher frequencies of extramedullary involvement, loss of the X/Y chromosome, and trisomy 22 (\u003cb\u003eOnline Resource 5\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePrognostic Impact of KIT Mutations in Pediatric Patients with CBF-AML\u003c/h2\u003e \u003cp\u003eAt a median follow-up of 44.7 months (range, 0.8\u0026ndash;109.9), the 5-year EFS, OS, and CIR among pediatric patients with \u003cem\u003eKIT\u003c/em\u003e mutations were 85.6% \u0026plusmn; 3.6%, 91.1% \u0026plusmn; 3.0%, and 7.0% \u0026plusmn; 2.8%, respectively. These outcomes did not significantly differ from those of \u003cem\u003eKIT\u003c/em\u003e wild-type patients (EFS, 77.9% \u0026plusmn; 3.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.177; OS, 85.5% \u0026plusmn; 2.8%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.377; CIR, 11.6% \u0026plusmn; 2.6%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.303) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Subgroup analyses further confirmed that the presence or location of \u003cem\u003eKIT\u003c/em\u003e mutations did not significantly influence prognosis: patients with exon 17 mutations had comparable 5-year EFS, OS, and CIR to those with other \u003cem\u003eKIT\u003c/em\u003e mutations or wild-type \u003cem\u003eKIT\u003c/em\u003e (EFS, 85.3% \u0026plusmn; 4.3% vs. 86.4% \u0026plusmn; 6.3% vs. 77.9% \u0026plusmn; 3.1%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.395; OS, 88.7% \u0026plusmn; 4.1% vs. 96.4% \u0026plusmn; 3.6% vs. 85.5% \u0026plusmn; 2.8%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.298; CIR, 5.1% \u0026plusmn; 2.9% vs. 10.8%\u0026plusmn; 5.9% vs. 11.6% \u0026plusmn; 2.6%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.425) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similarly, no significant survival differences were observed between \u003cem\u003eKIT\u003c/em\u003e-mutated and wild-type patients within either the \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003eor \u003cem\u003eCBFβ::MYH11\u003c/em\u003e fusion subgroups (\u003cb\u003eOnline Resources 6 and 7\u003c/b\u003e). Among pediatric patients with \u003cem\u003eKIT\u003c/em\u003e mutations, outcomes were also comparable between fusion subtypes (EFS, 87.0% \u0026plusmn; 3.9% vs. 80.4% \u0026plusmn; 8.8%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.313; OS, 90.5% \u0026plusmn; 6.4% vs. 91.4% \u0026plusmn; 3.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.326; CIR, 7.1% \u0026plusmn; 3.1% vs. 6.7% \u0026plusmn; 6.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.936) (\u003cb\u003eOnline Resource 8\u003c/b\u003e), and between those receiving FLAG-IDA versus DAE induction regimens (EFS, 85.9% \u0026plusmn; 4.0% vs. 84.2% \u0026plusmn; 8.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.773; OS, 91.6% \u0026plusmn; 3.3% vs. 88.9% \u0026plusmn; 7.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.706; CIR, 8.4% \u0026plusmn; 3.3% vs. 0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.255) (\u003cb\u003eOnline Resource 9\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence and Prognostic Impact of FLT3-ITD Mutations​​\u003c/h2\u003e \u003cp\u003e \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations were identified in 16 (5.5%) pediatric patients with CBF-AML, with a higher incidence among those carrying \u003cem\u003eRUNX1::RUNX1T1\u003c/em\u003e (6.1%, 14/231) than in \u003cem\u003eCBFβ::MYH11\u003c/em\u003e cases (3.4%, 2/58). No significant differences in baseline characteristics were observed between \u003cem\u003eFLT3-\u003c/em\u003eITD\u0026ndash;positive and \u0026ndash;negative patients (\u003cb\u003eOnline Resource 10).\u003c/b\u003e Among the 16 CBF-AML patients harboring \u003cem\u003eFLT3\u003c/em\u003e-ITD, two discontinued treatment after the first induction course, and one died from infectious complications following the second induction. Of the 13 evaluable patients, four underwent hematopoietic stem cell transplantation (HSCT) during their first complete remission (CR1), and all remained event-free. The remaining nine patients received consolidation chemotherapy alone, with only one relapse and eight event-free survivors. Outcomes in \u003cem\u003eFLT3\u003c/em\u003e-ITD\u0026ndash;positive patients were not inferior to those without this mutation, with comparable 5-year EFS (75.0% \u0026plusmn; 10.8% vs. 80.8% \u0026plusmn; 2.5%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.438), OS (87.1% \u0026plusmn; 8.6% vs. 86.4% \u0026plusmn; 2.4%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.821), and CIR (8.4% \u0026plusmn; 3.3% vs. 10.1% \u0026plusmn; 2.0%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.800) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Although not statistically significant, patients with CBF-AML and \u003cem\u003eFLT3\u003c/em\u003e-ITD showed a trend toward better survival than those with non-CBF-AML harboring \u003cem\u003eFLT3\u003c/em\u003e-ITD (\u003cb\u003eOnline Resource 11\u003c/b\u003e). After excluding patients who underwent HSCT in CR1, CBF-AML patients with \u003cem\u003eFLT3-\u003c/em\u003eITD demonstrated significantly superior 5-year EFS (75.0% \u0026plusmn; 10.8% \u003cem\u003evs.\u003c/em\u003e 17.8% \u0026plusmn; 6.5%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) and OS (82.5% \u0026plusmn; 11.3% \u003cem\u003evs.\u003c/em\u003e 46.0% \u0026plusmn; 8.9%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.049), alongside a lower CIR (75.0% \u0026plusmn; 12.5% \u003cem\u003evs.\u003c/em\u003e 70.6% \u0026plusmn; 9.9%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005), compared to their non-CBF-AML counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRisk-Factor Analysis in Pediatric Patients with CBF-AML\u003c/h2\u003e \u003cp\u003eTen patients (3.5%) were excluded due to treatment discontinuation or transfer between institutions before the second induction, leaving 279 patients evaluable for the intention-to-treat (ITT) analysis. Univariate Cox regression was performed to evaluate the impact of baseline clinical and genetic variables, including age, sex, initial white blood cell (WBC) count, extramedullary involvement, fusion type \u003cem\u003e(RUNX1::RUNX1T1\u003c/em\u003e or \u003cem\u003eCBFβ::MYH11\u003c/em\u003e), additional cytogenetic abnormalities (\u0026minus;\u0026thinsp;X/\u0026minus;Y), and induction regimen (FLAG-IDA \u003cem\u003evs.\u003c/em\u003e DAE), on long-term survival. None of these factors were significantly associated with long-term outcomes (\u003cb\u003eOnline Resource 12\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, MRD status at TP2 emerged as a powerful prognostic indicator. Among 268 patients with available MRD data at TP2, those exhibiting MFC-MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.1% experienced significantly inferior 5-year EFS (70.1% \u0026plusmn; 7.3% \u003cem\u003evs.\u003c/em\u003e 87.3% \u0026plusmn; 2.3%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and OS (74.2% \u0026plusmn; 7.1% \u003cem\u003evs.\u003c/em\u003e 92.1% \u0026plusmn; 2.0%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and higher 5-year CIR (25.9% \u0026plusmn; 7.1% \u003cem\u003evs.\u003c/em\u003e 7.3% \u0026plusmn; 1.9%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), compared with those who were MRD-negative (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Similarly, molecular MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.1% was associated with worse EFS (75.6% \u0026plusmn; 4.6% \u003cem\u003evs.\u003c/em\u003e 89.8% \u0026plusmn; 2.4%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), OS (80.8% \u0026plusmn; 4.4% \u003cem\u003evs.\u003c/em\u003e 92.9% \u0026plusmn; 2.2%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003), and higher CIR (20.1% \u0026plusmn; 4.4% \u003cem\u003evs.\u003c/em\u003e 4.7% \u0026plusmn; 1.7%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis large multicenter analysis of pediatric patients with CBF-AML uniformly treated according to the C-HUANAN-AML-15 protocol revealed that the prognostic significance of receptor tyrosine kinase (RTK) mutations, specifically in \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3-ITD\u003c/em\u003e, is substantially attenuated in the context of intensive chemotherapy. In contrast, MRD after induction emerged as the dominant predictor of clinical outcomes, underscoring the value of dynamic risk assessment over static genetic profiling in this population.\u003c/p\u003e \u003cp\u003eConsistent with prior reports, \u003cem\u003eKIT\u003c/em\u003e mutations were present in approximately one-third of pediatric patients with CBF-AML, with exon 17 alterations being most prevalent [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The prognostic implications of \u003cem\u003eKIT\u003c/em\u003e mutations in pediatric CBF-AML remain debated. Historically, \u003cem\u003eKIT\u003c/em\u003e exon 17 mutations\u0026mdash;particularly D816 and N822\u0026mdash;have been associated with higher relapse risk [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, conflicting evidence exists, with several reports failing to confirm an independent adverse prognostic impact [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Functional analyses further suggest that the clinical behavior of \u003cem\u003eKIT\u003c/em\u003e mutations may depend on the mutation class (e.g., gain-of-function vs. kinase domain alterations) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, KIT mutations, regardless of subtype or fusion partner, did not significantly affect EFS, OS, or CIR. This lack of prognostic influence may stem from intensified frontline therapy, predominantly FLAG-IDA, which provides superior antileukemic activity compared to conventional \u0026ldquo;7\u0026thinsp;+\u0026thinsp;3\u0026rdquo; regimens. Supporting this, adult CBF-AML studies have shown that fludarabine-containing protocols, such as FLAG-IDA or FLAG combined with gemtuzumab ozogamicin (GO), can mitigate the negative effects of \u003cem\u003eKIT\u003c/em\u003e mutations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Our results align with this, suggesting that uniform FLAG-IDA treatment likely neutralizes \u003cem\u003eKIT\u003c/em\u003e-driven leukemogenesis. Discrepancies across studies may be attributable to variations in sample size, consolidation regimens, and MRD integration, as noted in recent reviews [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations were infrequent in our cohort (5.5%), consistent with their lower prevalence in CBF-AML than in other AML subtypes [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The prognostic relevance of \u003cem\u003eFLT3\u003c/em\u003e-ITD in CBF-AML appears to differ between adults and children. In adults, \u003cem\u003eFLT3\u003c/em\u003e-ITD confers inferior outcomes and challenges favorable-risk classification, though HSCT in CR1 yields limited benefit [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In contrast, pediatric studies indicate minimal impact. For example, the Japanese Childhood AML Cooperative Study Group found that \u003cem\u003eKIT\u003c/em\u003e\u0026mdash;but not \u003cem\u003eFLT3\u003c/em\u003e-ITD\u0026mdash;was strongly associated with relapse in t(8;21) cases [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], a finding echoed in European and US cooperative series [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eFLT3\u003c/em\u003e-ITD did not confer an adverse impact on survival in pediatric CBF-AML. Interestingly, the outcomes for CBF-AML patients harboring \u003cem\u003eFLT3\u003c/em\u003e-ITD were at least comparable and possibly superior to those of non-CBF-AML patients with \u003cem\u003eFLT3\u003c/em\u003e-ITD. When patients undergoing HSCT in CR1 were excluded, CBF-AML patients with \u003cem\u003eFLT3\u003c/em\u003e-ITD showed significantly higher EFS and OS and lower CIR than their non-CBF-AML counterparts, suggesting that CBF cytogenetics may buffer the oncogenic effects of \u003cem\u003eFLT3\u003c/em\u003e-ITD. Among 13 evaluable patients with \u003cem\u003eFLT3\u003c/em\u003e-ITD, all four receiving CR1-HSCT remained event-free, while only one of nine on chemotherapy alone relapsed, suggesting that chemotherapy may be sufficient in this context and routine upfront HSCT may not be necessary. However, the small sample size (n\u0026thinsp;=\u0026thinsp;16) limits the statistical power; thus, larger prospective MRD-integrated studies are needed to confirm whether pediatric CBF-AML with \u003cem\u003eFLT3\u003c/em\u003e-ITD can safely avoid transplantation.\u003c/p\u003e \u003cp\u003eIn pediatric AML, MRD monitoring has revolutionized risk stratification, with post-induction levels guiding therapy intensification and reducing overtreatment [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Across both fusion subtypes, MRD after induction is the most robust predictor of outcome. Patients with MFC- or PCR-based MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.1% had significantly poor survival, consistent with multiple pediatric AML studies [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These findings reinforce that MRD is a stronger prognostic marker than baseline mutations and should guide post-remission therapy.\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, despite being one of the largest multicenter pediatric CBF-AML cohorts in China, the sample size of patients harboring FLT3-ITD mutations was small (n\u0026thinsp;=\u0026thinsp;16), limiting the statistical power to draw definitive conclusions regarding their prognostic significance. Second, the non-randomized assignment of patients to FLAG-IDA versus DAE induction regimens introduces potential selection bias. Third, although MRD was systematically assessed, inter-laboratory variability across participating centers may have affected the uniformity of detection thresholds. These limitations highlight the need for larger, prospective, and ideally international collaborative studies integrating genomic and MRD profiling to refine risk stratification and therapeutic strategies for pediatric CBF-AML.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings suggest that within the framework of contemporary intensive chemotherapy, the historically adverse prognosis associated with \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations in pediatric CBF-AML may be neutralized. MRD levels after induction remain the most powerful prognostic biomarker, emphasizing the need for dynamic response-based risk stratification in future clinical trials.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our deepest appreciation to the patients who donated samples. We also extend our sincere gratitude to the physicians from the participating institutions for providing clinical data. Chunfu Li served as the principal investigator of the C-HUANAN-AML-15 protocol, and Xiaoqun Feng was the chairperson of the C-HUANAN-AML working party.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCompeting Interests\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Fujian Provincial Clinical Research Center for Hematological Malignancies (Grant No. 2020Y2006) and the Natural Science Foundation of Fujian Province (Grant No. 2025J01122).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEthics Approval and Consent to Participate\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical Approval No obtained from the Fujian Medical University Union Hospital Institutional Review Board (Approval No.: 2025KY666), with a waiver of informed consent granted in accordance with the national regulations and the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConsent for Publication\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData Availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available upon request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConceptualization:\u0026nbsp;\u003c/strong\u003eY.Z., L.Yu, and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData curation:\u0026nbsp;\u003c/strong\u003eS.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C., and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormal analysis:\u003c/strong\u003e Y.Z. and L.Yu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding acquisition:\u003c/strong\u003e N.L.\u0026nbsp;and Y.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigation:\u003c/strong\u003e Y.Z., L.Yu, and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethodology:\u003c/strong\u003e Y.Z., L.Yu, and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProject administration:\u003c/strong\u003e Y.Z., L.Yu, and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResources:\u003c/strong\u003e S.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C., and L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSoftware:\u003c/strong\u003e Y.Z. and L.Yu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupervision:\u003c/strong\u003e L.Yang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation:\u003c/strong\u003e All authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVisualization:\u003c/strong\u003e Y.Z. and L.Yu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – original draft:\u003c/strong\u003e Y.Z. and L.Yu\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWriting – review \u0026amp; editing:\u003c/strong\u003e S.L., N.L., X.F., C.L., M.Z., H.M., H.J., X.H., H.W., H.X., C.C.,\u0026nbsp;L.Yang\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDuployez N, Marceau-Renaut A, Boissel N et al (2016) Comprehensive mutational profiling of core binding factor acute myeloid leukemia. 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Am J Hematol 100(Suppl 2):5\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/ajh.27482\u003c/span\u003e\u003cspan address=\"10.1002/ajh.27482\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Pediatric acute myeloid leukemia, Core-binding factor acute myeloid leukemia, KIT mutations, FLT3 internal tandem duplication, Measurable residual disease","lastPublishedDoi":"10.21203/rs.3.rs-8473547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8473547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough core-binding factor acute myeloid leukemia (CBF-AML) is generally considered a favorable-risk subtype in children, disease relapse remains a significant concern. The prognostic relevance of co-occurring mutations, particularly \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3-ITD\u003c/em\u003e, remains debatable, and treatment intensity may modulate their impact.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThis multicenter analysis included 289 children (\u0026lt;\u0026thinsp;14 years) with newly diagnosed CBF-AML enrolled in the C-HUANAN-AML-15 study (2015\u0026ndash;2023). \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3-ITD\u003c/em\u003e mutations were identified via cytogenetic analysis and targeted sequencing. Measurable residual disease (MRD) was evaluated by multiparameter flow cytometry (MFC) and quantitative polymerase chain reaction (PCR) following induction chemotherapy. Survival analyses were performed using Kaplan\u0026ndash;Meier and Cox regression methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eKIT\u003c/em\u003e mutations were detected in 103 patients (35.6%), predominantly involving exon 17 (69.9%), and were associated with extramedullary disease, sex chromosome loss, and trisomy 22. No significant differences in 5-year event-free survival (EFS), overall survival (OS), or cumulative incidence of relapse (CIR) were observed between patients with and without \u003cem\u003eKIT\u003c/em\u003e mutations. \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations (5.5% of patients) did not adversely affect outcomes. Neither mutation independently predicted survival. MRD positivity (MFC-MRD\u0026thinsp;\u0026ge;\u0026thinsp;0.1%) after the second induction cycle strongly predicted inferior EFS and OS and higher CIR, with corresponding results observed for molecular MRD and parallel findings for PCR-based MRD.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eIn this large multicenter cohort, \u003cem\u003eKIT\u003c/em\u003e and \u003cem\u003eFLT3\u003c/em\u003e-ITD mutations did not adversely affect the prognosis of pediatric CBF-AML treated according to the C-HUANAN-AML-15 protocol. MRD after induction was the most powerful predictor of relapse and survival, underscoring its importance for risk stratification in future pediatric AML trials.\u003c/p\u003e","manuscriptTitle":"KIT and FLT3-ITD Mutations Do Not Predict Outcomes in Pediatric Core-Binding Factor Acute Myeloid Leukemia: Findings from the C-HUANAN-AML-15 Multicenter Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 05:50:36","doi":"10.21203/rs.3.rs-8473547/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-10T15:56:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T17:57:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-06T12:43:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87988375335422810539522783597405771887","date":"2026-03-24T08:32:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"258633361518489734426598028961033146553","date":"2026-03-24T07:03:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-07T01:20:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-02T13:21:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-02T13:17:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Annals of Hematology","date":"2025-12-29T13:30:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"annals-of-hematology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"aohe","sideBox":"Learn more about [Annals of Hematology](http://link.springer.com/journal/277)","snPcode":"277","submissionUrl":"https://submission.nature.com/new-submission/277/3","title":"Annals of Hematology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4e3dd022-00c9-4f56-a5ea-0abd01ad7b51","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-10T15:56:44+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T11:08:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 05:50:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8473547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8473547","identity":"rs-8473547","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}