The Clinical Relevance of RAS Pathway Gene Mutations in Pediatric B-Cell Acute Lymphoblastic Leukemia

doi:10.21203/rs.3.rs-8082043/v1

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The Clinical Relevance of RAS Pathway Gene Mutations in Pediatric B-Cell Acute Lymphoblastic Leukemia

https://doi.org/10.21203/rs.3.rs-8082043/v1

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Objective

High-throughput sequencing has elucidated the genetic landscape of B-cell acute lymphoblastic leukemia (B-ALL). Notably, FLT3 and RAS pathway gene mutations represent a significant proportion of pediatric cases. Our study explores whether these patients have distinct clinical features and prognostic outcomes.

Methods

We retrospectively analyzed 562 pediatric B-ALL cases from the CCCG-ALL-2015 cohort using whole transcriptome sequencing to assess the clinical features of FLT3 mutations, RAS pathway alterations (NRAS, KRAS, PTPN11, NF1), and their cooperative effects.

Results

Among the cohort, 224 patients (39.86%) carried at least one RAS pathway alterations (NRAS, n = 120; KRAS, n = 109; PTPN11, n = 57; NF1, n = 11), while 82 patients (14.59%) had FLT3 mutations. Strikingly, 6 of 11 NF1-mutated patients harbored concurrent FLT3 mutations. A significant correlation was observed between FLT3 and NF1 mutations (Phi coefficient = 0.16, χ² = 11.518, p < 0.0001), NRAS and KRAS mutations (Phi coefficient = 0.17, χ² = 15.713, p < 0.0001). Patients with FLT3/NF1 co-mutations exhibited a higher frequency of abnormal karyotypes. Survival analysis revealed that these patients had significantly poorer overall survival (OS) and event-free survival (EFS) (p < 0.05), particularly when compared to those without KMT2A rearrangements (p = 0.0022 and p = 0.0026, respectively). Collectively, RAS pathway alterations were not significantly associated with inferior OS or EFS. Multivariate Cox regression analysis confirmed that FLT3/NF1 co-mutations, as a distinct molecular subtype, were independently associated with inferior OS (HR: 18.663, 95% CI: 2.203–158.106; p = 0.007) and EFS (HR: 4.986, 95% CI: 1.167–21.304; p = 0.03). PTPN11 mutations (HR 2.67, 95% CI 1.41–5.04) and FLT3/NF1 co-mutations (HR 7.08, 95% CI 1.22–41.09) also showed significant associations with Day 19 MRD ≥ 0.1% and Day 46 MRD ≥ 0.01%, respectively.

Conclusion

Our findings demonstrate that FLT3/NF1 co-mutations, but not RAS pathway mutations, defined high-risk pediatric B-ALL with poor outcomes.

Trial registration

: The study was conducted with approval from the Institutional Review Board of Children's Hospital of Soochow University (Approval number: 2019KS006).

B-Cell acute lymphocytic leukemia

FLT3

NF1

RAS pathway

B-cell acute lymphoblastic leukemia (B-ALL), the most common childhood malignancy, is characterized by the clonal expansion of immature B cells, leading to impaired normal hematopoiesis (1). Over the past two decades, risk-stratified treatment strategies, including intensified chemotherapy regimens and minimal/measurable residual disease (MRD)-guided therapy, have significantly improved clinical outcomes in B-ALL (2, 3). Although the overall survival rate for pediatric B-ALL now exceeds 90%, high-risk patients and those experiencing relapse continue to face treatment failure and dismal prognoses (4–6). Consequently, elucidating novel molecular mechanisms and identifying therapeutic targets remain critical for improving outcomes in refractory or relapsed B-ALL.

The advent of whole transcriptome sequencing (WTS) has revolutionized the molecular classification of B-ALL, enabling the discovery of novel subtypes and refining risk stratification beyond conventional criteria. The FMS-like tyrosine kinase 3 (FLT3) gene, located on chromosome 13q12, encodes a transmembrane RTK that plays a crucial role in hematopoiesis by serving as a key cytokine receptor (7). FLT3 mutations occur in approximately 5–10% of pediatric B-ALL cases (8, 9). While the prognostic significance of FLT3 mutations, particularly FLT3-ITD (Internal Tandem Duplication), is a well-established oncogenic driver in acute myeloid leukemia (AML), their impact on B-ALL outcomes remains unclear (7, 10). FLT3 functions upstream of the RAS-ERK pathway, mediating extracellular signal transduction and activating downstream cascades, including the RAS pathway, to regulate cellular processes. Notably, RAS pathway mutations (e.g., NRAS, KRAS, PTPN11, NF1) are present in about 20–36% of pediatric B-ALL cases (8, 11–13). Among these, NRAS and KRAS mutations are more frequent in pediatric than adult B-ALL, yet—like FLT3 mutations—they lack significant prognostic relevance (14, 15).

Earlier research on B-ALL has largely focused on single-gene alterations or fusion genes, leaving the clinical implications of combinatorial mutational patterns relatively unexplored. While growing evidence indicates that certain co-mutational signatures—particularly IKZF1 alterations coexisting with CDKN2A/B, PAX5, or PAR1 deletions—demonstrate robust correlations with unfavorable clinical outcomes (16, 17). Previous studies have primarily focused on NRAS/KRAS interactions or common mutational subtypes involving RAS or FLT3. Notably, FLT3 and RAS (NRAS or KRAS) mutations exhibit significant mutual exclusivity in hyperdiploid B-ALL (8, 18, 19). Subtype-specific analyses have further revealed distinct mutational patterns: FLT3, KRAS, and NRAS mutations are preferentially associated with hyperdiploid B-ALL, while NF1 mutations predominantly occur in hypodiploid subtypes (20, 21). Given the high prevalence and heterogeneity of FLT3 and RAS pathway alterations, we sought to systematically investigate their co-mutational patterns and clinical significance in pediatric B-ALL (22, 23).

In this study, we performed WTS on 562 pediatric patients with newly diagnosed B-ALL to investigate the co-occurrence of FLT3 mutations and RAS pathway alterations (NRAS, KRAS, NF1, and PTPN11), as well as their associated clinical characteristics and prognostic implications.

Data acquisition

This retrospective study was conducted with approval from the Institutional Review Board of Children's Hospital of Soochow University. Patient genetic profiles and clinical data were systematically extracted from the hospital's centralized electronic medical records database. In strict adherence to the ethical principles outlined in the Declaration of Helsinki, written informed consent was obtained from all study participants or their legal guardians prior to treatment initiation, which included provisions for biological specimen collection and subsequent research utilization.

Patients

In this retrospective cohort study, we initially screened 1,341 pediatric patients (aged 1–18 years) with newly diagnosed acute lymphoblastic leukemia (ALL) who underwent whole transcriptome sequencing (WTS) under the CCCG-ALL-2015 protocols between July 2015 and June 2020. After applying stringent inclusion criteria, 562 patients qualified for final analysis. Exclusion criteria resulted in the removal of 779 cases, comprising: incomplete WTS data (n = 707), T-cell lineage ALL (n = 51), younger than 1 age (n = 7), and loss to follow-up (n = 21).

Statistical analysis

All statistical analyses were performed using IBM SPSS Statistics (version 29.0.1.0) and R (version 4.2.2; R Foundation for Statistical Computing) with the following essential packages: survival for survival analyses, vcd for categorical data visualization, dplyr for data manipulation, and ggplot2 for graphical representations. Overall survival (OS) was calculated from the date of B-ALL diagnosis until death from any cause, with living patients censored at their last follow-up date.

Event-free survival (EFS) was measured from diagnosis to the first occurrence of: disease relapse, death from any cause or treatment failure with event-free patients censored at last follow-up. Categorical variables were analyzed using χ² tests or Fisher's exact tests, as appropriate based on expected cell frequencies. Survival curves were generated using the Kaplan-Meier method, with between-group differences assessed by log-rank tests. Multivariable Cox proportional hazards regression models were constructed to evaluate independent prognostic factors, utilizing both backward and forward stepwise selection based on likelihood ratio criteria (α = 0.05 for entry/removal). All statistical tests were two-sided, with p-values < 0.05 considered statistically significant.

From an initial cohort of 1,341 patients, we enrolled 562 pediatric B-ALL patients who completed WTS at diagnosis under the CCCG-ALL-2015 protocol. As shown in Supplemental Data table S1, the study population had a median age of 4.8 years (range: 3.2–7.9 years) with a male predominance (60.14% male vs 39.86% female). With a median follow-up of 2.67 years (IQR: 1.53–3.80 years), we identified RAS pathway mutations in 224 patients (39.86%), including NRAS (n = 120), KRAS (n = 109), PTPN11 (n = 57), and NF1 (n = 11) variants. Additionally, FLT3 mutations were detected in 82 patients (14.59%).

Co-occurrence analysis between FLT3 and RAS pathway genes

As illustrated in Fig. 1, nearly half of the patients with FLT3 mutations also harbored concurrent alterations in RAS pathway genes. To assess potential associations between FLT3 mutations and RAS pathway alterations, we performed Pearson residual analysis and chi-square tests. Notably, as shown in Fig. 2, co-mutation of FLT3 and NF1 occurred more frequently than expected by chance, as evidenced by a high Pearson residual (3.5). Statistical analysis confirmed significant positive associations between FLT3 and NF1 mutations (Phi coefficient = 0.16, χ² = 11.518, p < 0.0001), and between NRAS and KRAS (Phi coefficient = 0.17, χ² = 15.713, p < 0.0001), indicating non-independence and a strong tendency for co-occurrence. In contrast, no significant correlation was observed between FLT3 mutations and alterations in NRAS, KRAS, or PTPN11 (Supplemental Data Table S2).

Clinical characteristics of the enrolled patients

We analyzed the clinical and cytogenetic characteristics of patients with RAS pathway and FLT3 mutations (Supplemental Data Table S3) and stratified the 562 cases into eight non-overlapping molecular subtypes based on WTS data. Patients harboring RAS pathway alterations exhibited a significantly higher prevalence of hyperdiploidy and DUX4 rearrangement (all p < 0.05), whereas the frequencies of ETV6::RUNX1, BCR::ABL1, and TCF3::PBX1 subtypes were markedly reduced compared to those without RAS pathway mutations (all p < 0.05). Among FLT3-mutated cases, hyperdiploidy was more common (p < 0.001), while TCF3::PBX1 and ETV6::RUNX1 subtypes were underrepresented (p < 0.05). Compared to wild-type patients, those with NRAS/KRAS co-mutations showed no significant differences in clinical characteristics, except for a markedly higher prevalence of hyperdiploidy (p < 0.001) and significantly lower frequency of ETV6-RUNX1 fusion (p = 0.03).

MRD was assessed on days 19 and 46 post-treatment. As shown in Supplemental Data table S3, PTPN11-mutated patients showed a significantly higher rate of MRD ≥ 0.1% than those without PTPN11 mutations (p < 0.001) at day 19. By day 46, two of six cases (33.33%) with concurrent FLT3 and NF1 mutations had MRD ≥ 0.1%, a proportion significantly elevated compared to patients lacking these co-mutations (1.55%, p = 0.005).

While not reaching statistical significance (p = 0.061), we observed that 4 of 6 (66.7%) patients with FLT3/NF1 co-mutations exhibited abnormal karyotypes. In contrast, all 5 patients with NF1 mutations alone showed normal karyotypes (Table 1). Among the FLT3-mutated cases, molecular characterization revealed that 5 patients harbored FLT3-tyrosine kinase domain mutations (TKD), while 1 case presented with a juxtamembrane region insertion mutation-both classified as non-ITD domain alterations. Additionally, all patients with FLT3/NF1 co-mutations were classified as B-other subtype and could not be assigned to any current molecular subtype of B-ALL.

Overall Survival

The overall 3-year OS rate for all enrolled patients was 97.89% ± 0.69%. Supplemental Data Table S4 demonstrates that both initial WBC (white blood cell) count ≥ 100×10⁹/L and Day 46 MRD ≥ 0.01% were significantly associated with inferior OS (p = 0.0072 and p = 0.04, respectively). Kaplan-Meier analysis of RAS pathway mutations and FLT3 mutations individually showed no significant prognostic impact (Supplemental Data Table S4).

Strikingly, patients with concurrent FLT3 and NF1 mutations exhibited markedly worse outcomes, with a 3-year OS of 75.0% ± 21.65% compared to 98.09% ± 0.64% in wild-type patients (p < 0.01; Fig. 1). This survival difference reached statistical significance (p = 0.003), while no other co-occurring mutations showed significant prognostic value (Supplemental Data Table S5).

Comparative survival analysis among molecular subtypes revealed distinct prognostic patterns (Fig. 1). Patients with FLT3/NF1 co-mutations demonstrated comparable OS to KMT2Ar subtype (p = 0.30), but exhibited significantly worse outcomes compared to non-KMT2Ar subtypes (p = 0.0022). Univariate analysis identified five clinical variables significantly associated with reduced OS (Supplemental Data Table S6). Subsequent multivariate Cox proportional hazards regression analysis established three independent predictors of shorter OS: FLT3/NF1 co-mutations (HR = 18.66, 95% CI = 2.20-158.11, p = 0.007), WBC ≥ 100×10⁹/L at diagnosis (HR = 6.83, 95% CI = 1.70-27.48, p = 0.007), and MEF2D mutated subtype (HR = 21.72, 95% CI = 2.57–183.50, p = 0.005) (Fig. 2).

Event-free Survival

The 3-year EFS for all enrolled patients was 89.86% ± 1.53% (Supplemental Data Fig. 1). Survival analysis identified several significant prognostic factors: male gender (p = 0.0014), WBC ≥ 100×10⁹/L (p < 0.0001), and MRD ≥ 0.01% at both day 19 and day 46 (all p < 0.05) were all associated with inferior EFS (Supplemental Data Table S5). Molecular characterization revealed that FLT3/KRAS co-mutations and ETV6::RUNX1 fusion correlated with favorable outcomes, while KMT2Ar predicted poor prognosis (all p < 0.05, Supplemental Data Table S5).

As shown in Fig. 1, comparative analysis demonstrated significantly worse 2 year-EFS in patients with FLT3/NF1 co-mutations versus those without (75.0% vs 93.30% at 2 years; p = 0.0061). When compared to other B-ALL subtypes, FLT3/NF1 co-mutated cases showed inferior outcomes. Notably, the prognosis of FLT3/NF1 co-mutated patients was comparable to KMT2Ar cases (p = 0.86) but significantly worse than non-KMT2Ar patients (p = 0.002).

We subsequently conducted univariate and multivariate Cox regression analyses to evaluate prognostic factors (Supplemental Data Table S6, Fig. 2). Among seven variables initially considered for multivariate modeling, five demonstrated independent prognostic significance. The final multivariate model identified FLT3/NF1 co-mutations (HR = 4.99, 95% CI = 1.17–21.30, p = 0.03) and KMT2Ar (HR = 3.92, 95% CI = 1.47–10.47, p = 0.006) as significant predictors of reduced EFS, along with WBC ≥ 100×10⁹/L and male gender (Fig. 2). However, apart from NF1 and KRAS, other genes in the RAS pathway, when co-occurring as mutated genes with FLT3, were not significantly associated with either EFS or OS probabilities using Kaplan-Meier methods based on log rank test (Supplemental Data Table S5).

Univariate and multivariable analysis of risk factors on MRD

Table 1 displays the distribution of MRD positivity rates at different threshold levels on Day 19 and Day 46. As shown in Fig. 2, multivariable analyses revealed that ZNF384 (OR 4.64, 95% CI 1.29–16.72), Ph-like (OR 7.28, 95% CI 1.84–28.72), KMT2Ar (OR 4.98, 95% CI 1.7-14.61) subtypes were independent risk factors for Day 19 MRD ≥ 1%. Notably, ETV6::RUNX1 subtype showed consistent protection across all MRD thresholds (≥ 1%, ≥ 0.1%, and ≥ 0.01%), in contrast to DUX4 which remained a risk factor at all levels (all p < 0.05). Additionally, hemoglobin levels (OR 0.99, 95% CI 0.98-1.00), PTPN11 mutations (OR 2.67, 95% CI 1.41–5.04), and Ph-like subtype (OR 9.91, 95% CI 1.98–49.48) were independently associated with Day 19 MRD ≥ 0.1%, while ZNF384 subtype (OR 5.98, 95% CI 1.27–28.13) was specifically associated with Day 19 MRD ≥ 0.01% (Fig. 2).

For Day 46 MRD analysis, limited case numbers restricted evaluation to the ≥ 0.01% threshold, where male sex (OR 2.51, 95% CI 1.06–5.95), FLT3/NF1 co-mutations (OR 7.08, 95% CI 1.22–41.09), and Ph-like subtype (OR 5.56, 95% CI 1.05–29.53) showed significant independent associations with MRD persistence in multivariable analysis (Fig. 2).

Genomic mutations in pediatric B-ALL play an increasingly critical role in defining molecular subtypes, refining risk stratification, and guiding therapeutic strategies (19). However, certain rare mutation subtypes or combinatorial mutational patterns in B-ALL—along with their clinical implications—remain incompletely characterized. In this cohort of 562 pediatric B-ALL patients, FLT3 mutations and alterations in the RAS pathway were detected in 82 (14.4%) and 224 (39.4%) cases, respectively. Notably, approximately 50% of the FLT3-mutated patients harbored concurrent RAS pathway alterations. Our result shows that RAS pathway mutations occurred in 39.86% cases in China, the frequency in our cohort was consistent with previous studies (24–26). Intriguingly, the co-occurrence between FLT3 and NF1 mutations was statistically significant and specific, unlike other RAS and FLT3 mutation pairs. Furthermore, we identified a significant positive correlation between FLT3 and NF1 co-mutations and inferior prognosis. Multivariate analysis confirmed that FLT3-NF1 co-mutations independently predicted adverse effects on both EFS and OS. However, aside from FLT3/NF1 co-mutations, neither FLT3, NF1, nor other RAS pathway genes—either alone or in combination—showed a significant prognostic impact in our analysis.

Given the prognostic significance of the FLT3/NF1 co-mutation, we sought to contextualize it within the broader landscape of FLT3 biology. As mentioned above, FLT3 encodes a class III receptor tyrosine kinase that belongs to the RTK signaling pathway family and plays essential roles in normal and malignant hematopoiesis (27). FLT3 kinase is typically activated by TKD point mutations or ITDs, with both types showing comparable prevalence in pediatric B-ALL (21, 28, 29). In our co-mutated subgroup, TKD point mutations were predominant (5 of 6 cases). Although FLT3 inhibitors have shown success in AML (30), their efficacy varies considerably depending on the specific FLT3 mutation type. Supporting this notion, Gutierrez-Camino et al. (21) identified a new activating mutation of TKD that exhibited oncogenic properties and resistance to sorafenib. Therefore, targeted therapies for FLT3-mutated B-ALL are a promising yet underdeveloped area, warranting further investigation (28).

In contrast to FLT3, the role of NF1 mutations in pediatric B-ALL remains poorly characterized, and their clinical implications are largely unexplored (8, 20). The NF1 gene encodes neurofibromin, a critical negative regulator of the RAS/MAPK signaling pathway. Loss of NF1 function leads to constitutive RAS-GTP activation and hyperstimulation of the RAS-ERK pathway (15). While NF1 alterations are established drivers in other malignancies, including juvenile myelomonocytic leukemia and AML (31, 32), they are rare in pediatric B-ALL (1.9% in our cohort) and have been rarely studied in this context. Critically, although NF1 mutation alone showed no independent prognostic impact in our analysis—suggesting it is not a canonical driver in B-ALL—our integrated findings posit that it orchestrates 'latent synergies' with concurrent FLT3 mutations. This synergies between NF1 and other genes or signaling pathway has also been established in other malignancies (33, 34). We propose that this collaboration drives pathogenesis and adverse outcomes, particularly relapse and drug resistance. Furthermore, we observed a high frequency of karyotype abnormalities (5 of 6 cases, 83.3%) in the FLT3/NF1 co-mutated subgroup, albeit without a consistent pattern. This aligns with reports linking NF1 alterations to complex karyotypes in myeloid malignancies (35–37). The high frequency of karyotype abnormalities in these cases led us to propose that FLT3 and NF1 mutations collectively impair DNA repair and disrupt cell cycle regulation, potentially explaining the poor outcomes in these co-mutated cases.

Beyond genetic subtypes, MRD remains a critical independent prognostic factor in pediatric B-ALL, with MRD-directed strategies significantly improving clinical outcomes (38). In our study, we systematically explored risk factors associated with MRD positivity (defined as ≥ 0.1% or ≥ 0.01%). The PTPN11 gene encodes a key protein tyrosine phosphatase that regulates multiple cellular processes including proliferation, differentiation, and oncogenic transformation through its phosphatase activity. In B-ALL, gain-of-function PTPN11 mutations lead to constitutive phosphatase activation, promoting leukemic cell proliferation and facilitating the transition from pre-leukemic clones to overt leukemia (39). Notably, while PTPN11 mutations did not significantly impact overall survival, they were associated with a 2.67-fold increased risk of Day 19 MRD ≥ 0.1% (OR = 2.67, 95%CI:1.32–5.41, p = 0.006), consistent with its role in enhancing treatment resistance through sustained proliferative signaling.

B-ALL is characterized by significant heterogeneity, particularly in its genetic subtypes (driven by distinct initiating events), genomic profiles, and clonal architecture. The heterogeneity of the clonal structure is evident in our study. these FLT3 mutations exhibited a broad spectrum of variant allele frequencies ranging from approximately 2% to 30%, indicating substantial clonal heterogeneity within this genetic subgroup, where leukemia cells frequently harbor more than one mutation, making co-mutations a common phenomenon. Despite the limited cohort size precludes a definitive determination of whether FLT3/NF1 co-mutations reside within the same or distinct subclones in pediatric B-ALL, their independent impact on adverse prognosis is conclusively established. Further research into this heterogeneity is crucial for achieving precision medicine, which aims to tailor individualized treatment strategies based on each patient's unique genetic profile and clonal structure. Current risk-stratified therapy draws primarily upon genetic subtypes and other clinical features identified at initial diagnosis. Future strategies, however, must integrate the complexity of clonal architecture and the presence of specific subclones. This will enable preemptive intervention against subclones with poor prognostic potential and facilitate their dynamic monitoring. Consequently, for patients with FLT3/NF1 co-mutations, the exploratory use of matched targeted agents—such as FLT3 inhibitors or MEK inhibitors to counter the downstream effects of NF1 loss—represents a promising therapeutic strategy.

Our findings demonstrate that FLT3 frequently co-occurs with RAS pathway gene mutations and FLT3/NF1 co-mutations in particular correlate with inferior clinical outcomes. Our findings also provide a potential strategy for this kind of patients integrating FLT3 or MEK inhibitor into the current treatment structure like TKI for BCR::ABL patients in future.

B-ALL

B-cell Acute Lymphoblastic Leukemia

WTS

Whole Transcriptome Sequencing

MRD

Minimal Residual Disease

TKD

Tyrosine Kinase Domain

ITD

Internal Tandem Duplication

Overall Survival

WBC

White Blood Cell

Hazard Ratio

95% CI

95% Confidence Interval

EFS

Event-Free Survival

AML

Acute Myeloid Leukemia

Odds Ratio

TKI

Tyrosine Kinase Inhibitor

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by Children's Hospital of Soochow University (Approve nubmber: 2019KS006). The studies were conducted in compliance with local laws and institutional regulations. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

Consent for publication

Not applicable

Availability of data and materials

The data supporting this study's findings are available on request from the corresponding author. However, due to privacy or ethical restrictions, they are not publicly available.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interests.

Funding

This study was supported by the National Key Research and Development Program of China (No. 2022YFC2502700), the National Natural Science Foundation of China (grant: 82170218, 82470221, 82103917, 82400264，82470127 and 82200177), Suzhou Key project (DZXYJ202305, SZS201615, SZS2023014, SKY2022012).

Author contributions

C-XY provided contributions to conducting the statistical analysis, research design, and drafting the article. Z-YP, H-YX, P-J and L-ZH performed data management and bioinformatics analysis. C-XY, L-YZ and H-SY edited and revised the article. All authors read and approved the final version of the manuscript.

Acknowledgments

Thanks to all authors for their contributions to this manuscript.

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69(1):7-34.DOI:10.3322/caac.21551.
Shah NN, Lee DW, Yates B, Yuan CM, Shalabi H, Martin S, et al. Long-Term Follow-Up of CD19-CAR T-Cell Therapy in Children and Young Adults With B-ALL. J Clin Oncol. 2021;39(15):1650-59.DOI:10.1200/jco.20.02262.
Hodder A, Mishra AK, Enshaei A, Baird S, Elbeshlawi I, Bonney D, et al. Blinatumomab for First-Line Treatment of Children and Young Persons With B-ALL. J Clin Oncol. 2024;42(8):907-14.DOI:10.1200/jco.23.01392.
Pieters R, de Groot-Kruseman H, Van der Velden V, Fiocco M, van den Berg H, de Bont E, et al. Successful Therapy Reduction and Intensification for Childhood Acute Lymphoblastic Leukemia Based on Minimal Residual Disease Monitoring: Study ALL10 From the Dutch Childhood Oncology Group. J Clin Oncol. 2016;34(22):2591-601.DOI:10.1200/jco.2015.64.6364.
Toft N, Birgens H, Abrahamsson J, Griškevičius L, Hallböök H, Heyman M, et al. Results of NOPHO ALL2008 treatment for patients aged 1-45 years with acute lymphoblastic leukemia. Leukemia. 2018;32(3):606-15.DOI:10.1038/leu.2017.265.
Pui CH. Precision medicine in acute lymphoblastic leukemia. Front Med. 2020;14(6):689-700.DOI:10.1007/s11684-020-0759-8.
Lo Schiavo F, Salvesi C, Jandoubi M, Pirini F, Garbetta J, Martinelli G, et al. Novel molecular mechanisms of FLT3 deregulation: from the acute myeloid leukemia experience to therapeutic insights in acute lymphoblastic leukemia. Mol Cancer. 2025;24(1):266.DOI:10.1186/s12943-025-02455-y.
Brady SW, Roberts KG, Gu Z, Shi L, Pounds S, Pei D, et al. The genomic landscape of pediatric acute lymphoblastic leukemia. Nat Genet. 2022;54(9):1376-89.DOI:10.1038/s41588-022-01159-z.
Biojone ER, Guido BC, Cavalcante LLM, Dos Santos Júnior ACM, de Pontes RM, Furtado FM, et al. Prevalence of FLT3 gene mutation and its expression in Brazilian pediatric B-ALL patients: clinical implications. Front Pediatr. 2024;12:1505060.DOI:10.3389/fped.2024.1505060.
Haage TR, Schraven B, Mougiakakos D, Fischer T. How ITD Insertion Sites Orchestrate the Biology and Disease of FLT3-ITD-Mutated Acute Myeloid Leukemia. Cancers (Basel). 2023;15(11).DOI:10.3390/cancers15112991.
Huang YJ, Liu HC, Jaing TH, Wu KH, Wang SC, Yen HJ, et al. RAS pathway mutation is an added-value biomarker in pediatric Philadelphia-negative B-cell acute lymphoblastic leukemia with IKZF1 deletions. Pediatr Blood Cancer. 2021;68(4):e28899.DOI:10.1002/pbc.28899.
Tran TH, Langlois S, Meloche C, Caron M, Saint-Onge P, Rouette A, et al. Whole-transcriptome analysis in acute lymphoblastic leukemia: a report from the DFCI ALL Consortium Protocol 16-001. Blood Adv. 2022;6(4):1329-41.DOI:10.1182/bloodadvances.2021005634.
Li X, Lin S, Liao N, Mai H, Long X, Liu L, et al. The RAS-signaling-pathway-mutation-related prognosis in B-cell acute lymphoblastic leukemia: A report from South China children's leukemia group. Hematol Oncol. 2024;42(3):e3265.DOI:10.1002/hon.3265.
Perentesis JP, Bhatia S, Boyle E, Shao Y, Shu XO, Steinbuch M, et al. RAS oncogene mutations and outcome of therapy for childhood acute lymphoblastic leukemia. Leukemia. 2004;18(4):685-92.DOI:10.1038/sj.leu.2403272.
Messina M, Chiaretti S, Wang J, Fedullo AL, Peragine N, Gianfelici V, et al. Prognostic and therapeutic role of targetable lesions in B-lineage acute lymphoblastic leukemia without recurrent fusion genes. Oncotarget. 2016;7(12):13886-901.DOI:10.18632/oncotarget.7356.
Felice MS, Rubio PL, Digiorge J, Barreda Frank M, Martínez CS, Guitter MR, et al. Impact of IKZF1 Deletions in the Prognosis of Childhood Acute Lymphoblastic Leukemia in Argentina. Cancers (Basel). 2022;14(13).DOI:10.3390/cancers14133283.
Wang'ondu RW, Ashcraft E, Chang TC, Roberts KG, Brady SW, Fan Y, et al. Heterogeneity of IKZF1 genomic alterations and risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia. Leukemia. 2025.DOI:10.1038/s41375-025-02633-3.
Armstrong SA, Mabon ME, Silverman LB, Li A, Gribben JG, Fox EA, et al. FLT3 mutations in childhood acute lymphoblastic leukemia. Blood. 2004;103(9):3544-6.DOI:10.1182/blood-2003-07-2441.
Li JF, Dai YT, Lilljebjörn H, Shen SH, Cui BW, Bai L, et al. Transcriptional landscape of B cell precursor acute lymphoblastic leukemia based on an international study of 1,223 cases. Proc Natl Acad Sci U S A. 2018;115(50):E11711-e20.DOI:10.1073/pnas.1814397115.
Ueno H, Yoshida K, Shiozawa Y, Nannya Y, Iijima-Yamashita Y, Kiyokawa N, et al. Landscape of driver mutations and their clinical impacts in pediatric B-cell precursor acute lymphoblastic leukemia. Blood Adv. 2020;4(20):5165-73.DOI:10.1182/bloodadvances.2019001307.
Gutierrez-Camino A, Richer C, Ouimet M, Fuchs C, Langlois S, Khater F, et al. Characterisation of FLT3 alterations in childhood acute lymphoblastic leukaemia. Br J Cancer. 2024;130(2):317-26.DOI:10.1038/s41416-023-02511-8.
Jerchel IS, Hoogkamer AQ, Ariës IM, Steeghs EMP, Boer JM, Besselink NJM, et al. RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia. Leukemia. 2018;32(4):931-40.DOI:10.1038/leu.2017.303.
Li Z, Zhao H, Yang W, Maillard M, Yoshimura S, Hsiao YC, et al. Molecular and pharmacological heterogeneity of ETV6::RUNX1 acute lymphoblastic leukemia. Nat Commun. 2025;16(1):1153.DOI:10.1038/s41467-025-56229-7.
Jerchel IS, Hoogkamer AQ, Ariës IM, Steeghs EMP, Boer JM, Besselink NJM, et al. RAS pathway mutations as a predictive biomarker for treatment adaptation in pediatric B-cell precursor acute lymphoblastic leukemia. Leukemia. 2018;32(4):931-40
Liang DC, Chen SH, Liu HC, Yang CP, Yeh TC, Jaing TH, et al. Mutational status of NRAS, KRAS, and PTPN11 genes is associated with genetic/cytogenetic features in children with B-precursor acute lymphoblastic leukemia. Pediatr Blood Cancer. 2018;65(2).DOI:10.1002/pbc.26786.
Al-Kzayer LFY, Saeed RM, Ghali HH, Tanaka M, Al-Jadiry MF, Faraj SA, et al. Comprehensive genetic analyses of childhood acute leukemia in Iraq using next-generation sequencing. Transl Pediatr. 2023;12(5):827-44.DOI:10.21037/tp-22-512.
Medina KL. Flt3 Signaling in B Lymphocyte Development and Humoral Immunity. Int J Mol Sci. 2022;23(13).DOI:10.3390/ijms23137289.
Zhang Y, Zhang Y, Wang F, Wang M, Liu H, Chen X, et al. The mutational spectrum of FLT3 gene in acute lymphoblastic leukemia is different from acute myeloid leukemia. Cancer Gene Ther. 2020;27(1-2):81-88.DOI:10.1038/s41417-019-0120-z.
Rücker FG, Du L, Luck TJ, Benner A, Krzykalla J, Gathmann I, et al. Molecular landscape and prognostic impact of FLT3-ITD insertion site in acute myeloid leukemia: RATIFY study results. Leukemia. 2022;36(1):90-99.DOI:10.1038/s41375-021-01323-0.
Wang ES, Goldberg AD, Tallman M, Walter RB, Karanes C, Sandhu K, et al. Crenolanib and Intensive Chemotherapy in Adults With Newly Diagnosed FLT3-Mutated AML. J Clin Oncol. 2024;42(15):1776-87.DOI:10.1200/jco.23.01061.
Kiuru M, Busam KJ. The NF1 gene in tumor syndromes and melanoma. Lab Invest. 2017;97(2):146-57.DOI:10.1038/labinvest.2016.142.
Karaconji T, Whist E, Jamieson RV, Flaherty MP, Grigg JRB. Neurofibromatosis Type 1: Review and Update on Emerging Therapies. The Asia-Pacific Journal of Ophthalmology. 2019;8(1)
Brundage ME, Tandon P, Eaves DW, Williams JP, Miller SJ, Hennigan RH, et al. MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1. Oncogene. 2014;33(49):5626-36.DOI:10.1038/onc.2013.506.
Sokol ES, Feng YX, Jin DX, Basudan A, Lee AV, Atkinson JM, et al. Loss of function of NF1 is a mechanism of acquired resistance to endocrine therapy in lobular breast cancer. Ann Oncol. 2019;30(1):115-23.DOI:10.1093/annonc/mdy497.
Kaburagi T, Yamato G, Shiba N, Yoshida K, Hara Y, Tabuchi K, et al. Clinical significance of RAS pathway alterations in pediatric acute myeloid leukemia. Haematologica. 2022;107(3):583-92.DOI:10.3324/haematol.2020.269431.
Safonov A, Nomakuchi TT, Chao E, Horton C, Dolinsky JS, Yussuf A, et al. A genotype-first approach identifies high incidence of NF1 pathogenic variants with distinct disease associations. Nat Commun. 2025;16(1):3121.DOI:10.1038/s41467-025-57077-1.
Tariq H, Loxas M, Alikhan MB, Gao J, Lu X, Chen QC, et al. Clinicopathologic Characteristics and Prognostic Profile of Chronic Myeloid Neoplasms With Somatic NF1 Mutations in Adult Patients. Eur J Haematol. 2025;115(1):46-56.DOI:10.1111/ejh.14419.
Sutton R, Shaw PJ, Venn NC, Law T, Dissanayake A, Kilo T, et al. Persistent MRD before and after allogeneic BMT predicts relapse in children with acute lymphoblastic leukaemia. Br J Haematol. 2015;168(3):395-404.DOI:10.1111/bjh.13142.
Bueno C, Tejedor JR, Bashford-Rogers R, González-Silva L, Valdés-Mas R, Agraz-Doblás A, et al. Natural history and cell of origin of TC F3-ZN F384 and PTPN11 mutations in monozygotic twins with concordant BCP-ALL. Blood. 2019;134(11):900-05.DOI:10.1182/blood.2019000893.

Table 1 is available in the Supplementary Files section

No competing interests reported.

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The Clinical Relevance of RAS Pathway Gene Mutations in Pediatric B-Cell Acute Lymphoblastic Leukemia

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