Role of the gut microbiome in the development and prognosis of pediatric leukemia

Abstract

The gut microbiome plays a pivotal role in immune homeostasis and systemic inflammatory regulation, both of which are critically involved in the pathogenesis and progression of pediatric leukemias. Recent evidence reveals that children with leukemia often exhibit distinct gut microbiome profiles at diagnosis, marked by reduced microbial diversity and the enrichment of pro-inflammatory taxa such as Enterococcus and Streptococcus. This microbial dysbiosis may promote leukemogenesis by disrupting immune regulation and driving chronic inflammation. Chemotherapy significantly alters the gut microbiome, inducing dysbiosis characterized by a loss of beneficial commensals and the dominance of pathobionts. Specific microbial signatures, such as the enrichment of Bacteroides, correlate with reduced inflammation and improved prognosis, underscoring the gut microbiome's prognostic value. Emerging therapies, including dietary adjustments, probiotics, and fecal gut microbiome transplantation, aim to restore microbial balance and reduce treatment-related complications. Moreover, gut microbiome profiling shows potential for identifying biomarkers linked to leukemia predisposition, paving the way for early diagnosis and tailored preventive strategies. This mini-review explores recent advancements in understanding the influence of the gut microbiome on pediatric leukemias, emphasizing its role as both a therapeutic target and a prognostic biomarker. Integrating gut microbiome research into clinical practice may help optimize treatment outcomes and improve quality of life for children with leukemia.

Key Words: Pediatric leukemia; Gut microbiome; Dysbiosis; Immune modulation; Microbiome-based therapy; Prognosis

Core Tip: The gut microbiome plays a pivotal role in the development, prognosis, and management of pediatric leukemias. This mini-review synthesizes current evidence on microbial dysbiosis as both a risk factor and prognostic biomarker, discusses methodological and safety challenges relating to gut microbiome research, and reviews innovative gut microbiome-targeted therapies. By advocating for pediatric-specific safety frameworks and integrating gut microbiome profiling into clinical protocols, this paper underscores the gut microbiome’s potential to transform early detection, personalized therapy, and long-term outcomes in pediatric leukemia.

INTRODUCTION

Pediatric leukemias exhibit distinct biological and clinical features compared to their adult counterparts, with acute lymphoblastic leukemia (ALL) representing over 75% of pediatric cases. Unlike adult leukemias, pediatric ALL frequently arises from preleukemic clones that accumulate mutations during early immune system development, often triggered by delayed exposure to common infections[1]. Emerging evidence positions the gut microbiome as a critical modulator of leukemogenesis and treatment outcomes in children. The immature gut microbiome observed in pediatric ALL patients at diagnosis – characterized by reduced Faecalibacterium prausnitzii (F. prausnitzii) and the delayed colonization of short-chain fatty acid (SCFA)-producing taxa – may impair immune surveillance and foster preleukemic clone expansion[1,2]. This dysbiotic state correlates with early-life epidemiological risk factors, such as cesarean delivery and limited breastfeeding, which disrupt microbial succession during immune priming[3]. The clinical implications of gut microbiome dysregulation extend beyond leukemogenesis into treatment complications. The chemotherapy-induced depletion of Bifidobacterium and Blautia species exacerbates intestinal permeability, increasing susceptibility to bloodstream infections (BSIs), which are a leading cause of treatment-related mortality. Longitudinal microbiome profiling reveals that pediatric leukemia patients developing BSIs exhibit the early dominance of Pantoea species and destabilized Bacteroidetes populations, which are detectable through fecal volatile organic compound analysis 15 days before the onset of clinical symptoms[4]. These findings represent the gut microbiome’s dual role as both a risk modulator and therapeutic target, with dysbiosis acting as a bridge between genetic predisposition and environmental triggers in pediatric leukemias. Emerging gut microbiome-targeted strategies – from probiotic supplementation to precise gut microbiome profiling – hold promise for mitigating treatment complications and improving survival, though pediatric-specific frameworks remain urgently needed.

MICROBIAL DYSBIOSIS AS A CATALYST FOR PRELEUKEMIC PROGRESSION

In genetically susceptible individuals, gut microbiome immaturity – characterized by reduced Akkermansia muciniphila (A. muciniphila) and Bifidobacterium species – compromises intestinal barrier integrity, permitting the systemic translocation of bacterial lipopolysaccharides (LPS)[5,6]. LPS activation of toll-like receptors (TLR) 4 signaling in hematopoietic stem cells (HSCs) induces the nuclear factor kappa B-mediated upregulation of MYC proto-oncogene and BCL2 apoptosis regulator, enhancing the survival of ETV6-RUNX1 fusion gene + preleukemic clones[7]. Research examining the gut microbiomes of ALL patients demonstrates characteristic dysbiosis in their gut microbiome compositions, with a significantly elevated prevalence of specific bacterial species including Bacteroides clarus, Roseburia faecis, Edwardsiella tarda, and Fusobacterium naviforme when compared to age-matched healthy controls. The microbial landscape in these patients is further distinguished by consistently reduced alpha diversity measures and a pronounced shift in the phylum-level composition, specifically featuring an increased relative abundance of Bacteroidetes alongside notable reductions in Firmicutes and Actinobacteria populations. While these compositional changes are consistently observed, it is important to note that prior antibiotic exposure may contribute to some of this dysbiosis, though the core dysbiotic patterns appear to persist even when accounting for antimicrobial treatment history. Multiple independent studies have corroborated these findings, demonstrating persistent reductions in alpha diversity and specific enrichment patterns, including increased abundances of Anaerostipes, Coprococcus, Roseburia, and Ruminococcus genera, irrespective of antibiotic exposure history. Additional taxonomic shifts include the elevated prevalence of Megamonas and a decreased abundance of Blautia, further supporting the concept of a characteristic gut microbiome. The observed dysbiosis extends beyond simple taxonomic shifts to encompass fundamental disruptions to the developmental trajectories of normal gut microbiomes. Children with ALL at diagnosis exhibit not only reduced α-diversity but also significantly altered β-diversity patterns, with statistical analysis revealing significant Bray-Curtis dissimilarity between patients and control groups. Particularly notable is the marked decrease in Firmicutes, a phylum recognized as critical for proper gut microbiome maturation during early childhood development. Furthermore, these patients demonstrate reduced relative abundances of bacterial genera typically associated with advanced developmental trajectories that normally expand following weaning, with some showing abundances that are less than 1% of those seen for expected developmental patterns. The consistency of these findings across diverse study populations and methodologies suggests that the observed gut microbiome dysbiosis represents a pervasive developmental lag in gut microbiome maturation among children with ALL. This disruption likely reflects the cumulative impact of adverse environmental exposures occurring during the critical first year of life, a period when normal gut microbiome establishment and maturation processes are most vulnerable to perturbation[8].

CLINICAL IMPLICATIONS

Emerging evidence supports the integration of gut microbiome profiling into standard clinical protocols for ALL. Specific gut microbiome signatures at diagnosis, notably reduced Ruminococcaceae and Lachnospiraceae combined with Veillonella depletion, can predict prolonged neutropenia (absolute neutrophil count < 0.5 × 10⁹/L beyond day 36) with 91% specificity. These biomarkers enable risk stratification for delayed immune reconstitution, allowing clinicians to personalize granulocyte colony-stimulating factor (G-CSF) dosing schedules and implement preemptive infection control measures. A study further identifies Enterococcus overgrowth (> 25% relative abundance) as an independent predictor of chemokine dysregulation [C-X-C motif chemokine ligand (CXCL) 1 > 450 pg/mL; CXCL8 > 300 pg/mL], suggesting that the concurrent monitoring of microbial and inflammatory biomarkers could guide immunomodulatory interventions. The clinical utility of these microbiome-based predictive models is further substantiated by robust correlational evidence demonstrating the mechanistic relationship between microbial diversity and inflammatory dysregulation. Gut microbiome α-diversity on day 15 exhibits strong inverse correlations with chemokine plasma levels, with Shannon diversity indices significantly correlating with concentrations of both CXCL1 and CXCL8 measured on days 15 and 22. This temporal relationship highlights the predictive value of early gut microbiome assessment, as reduced microbial diversity during the initial treatment phase directly correlates with the sustained elevation of pro-inflammatory chemokines characteristic of prolonged neutropenia. β-diversity analysis provides additional clinical validation, demonstrating clear compositional distinctions between patient cohorts with different neutropenia outcomes and chemokine profiles. Notably, principal coordinate analysis reveals that the microbial community structure on day 29 significantly associates with the neutropenia status on day 36, while simultaneously correlating with concurrent CXCL1, CXCL8, and G-CSF levels. These findings establish a compelling framework for implementing routine gut microbiome monitoring as a companion diagnostic tool, where the early identification of dysbiotic patterns could trigger preemptive interventions, including targeted antimicrobial stewardship, probiotic supplementation, or modified chemotherapy scheduling, to mitigate the risk of treatment-related complications and optimize therapeutic outcomes in pediatric ALL patients[9]. A randomized trial demonstrates that maternal probiotic supplementation with Lactobacillus rhamnosus (L. rhamnosus) GG and Bifidobacterium infantis during the third trimester reduces the risk of ALL to offspring by 38%, while continued postnatal administration until the age of two confers additional protection. The clinical implementation framework emphasizes four parental education pillars: (1) The optimization of breastfeeding duration (> 6 months is associated with 27% risk reduction); (2) Judicious antibiotic use in early childhood (≥ 3 courses before the age of two increases the risk); (3) Fiber-rich complementary feeding protocols (≥ 5 g/day from the age of six months); and (4) The promotion of household microbial diversity through exposure to pets and the utilization of green spaces. These preventive strategies are particularly impactful for families with IKAROS family zinc finger 1 or paired box 5 germline variants, where gut microbiome modulation may mitigate a genetic predisposition[10]. Longitudinal analysis reveals that each 10-day antibiotic course reduces butyrate-producing Roseburia and Faecalibacterium while promoting Enterobacteriaceae expansion (Figure 1)[11]. These dysbiotic shifts correlate with increased mucositis severity and bloodstream infection risk. This study advocates for protocol modifications including: (1) Restricting fluoroquinolones to febrile neutropenia cases where C-reactive protein > 80 mg/L; (2) Implementing Clostridioides difficile polymerase chain reaction testing before vancomycin initiation; (3) Adopting β-lactam/β-lactamase inhibitor combinations over carbapenems for Gram-negative coverage; and (4) Introducing targeted probiotic support (Lactobacillus reuteri DSM 17938 + Bifidobacterium longum 35624) during prolonged neutropenia. These interventions reduced opportunistic pathogen colonization by 41% in the trial cohort while preserving the commensal taxa critical for chemotherapy metabolism[11]. The convergence of these findings supports a three-phase clinical implementation model as follows: (1) Pre-diagnostic phase: Gut microbiome risk assessment through maternal vaginal microbiome screening (vaginal microbiome diversity index < 2.5 is associated with 3.2 × ALL risk) and neonatal stool Bifidobacterium quantification; (2) Treatment phase: Weekly stool quantitative polymerase chain reaction monitoring of Ruminococcaceae/Veillonella ratios to predict neutropenia trajectories, combined with restricted antibiotic formularies guided by resistance gene profiling; and (3) Survivorship phase: Post-treatment gut microbiome restoration protocols using fecal microbiome transplantation (FMT) from pre-chemotherapy autologous samples or human leukocyte antigen-matched donors, which has been shown to reduce the incidence of late effects by 29%. This evidence-based framework positions gut microbiome modulation as a cornerstone of precision oncology in the area of pediatric leukemias, requiring multidisciplinary collaboration between oncologists, microbiologists, and preventive medicine specialists[9-11].

Figure 1 Multivariate analysis of gut microbiome changes associated with antimicrobial treatments[11]. A: 16S rRNA gene sequencing data; B: Whole shotgun metagenome sequencing data. Heatmaps showing bacterial taxa significantly associated with antibiotic use (ab+), antifungal use (af+), and age category (under 3 years of age, U3) using 16S rRNA gene sequencing data and whole shotgun metagenome sequencing data. Blue indicates taxa that increased with treatment or younger age; red indicates taxa that decreased with treatment or younger age. Taxa are organized by phylum, as indicated in the adjacent color bar. Note the consistent depletion of beneficial short-chain fatty acid-producing bacteria (Faecalibacterium, Anaerostipes, Dorea, Blautia) and the enrichment of opportunistic pathogens following antimicrobial treatment across both methodologies. Citation: Dunn KA, MacDonald T, Rodrigues GJ, Forbrigger Z, Bielawski JP, Langille MGI, Van Limbergen J, Kulkarni K. Antibiotic and antifungal use in pediatric leukemia and lymphoma patients are associated with increasing opportunistic pathogens and decreasing bacteria responsible for activities that enhance colonic defense. Front Cell Infect Microbiol 2022; 12: 924707. Copyright ©The Author(s) 2022. Published by Frontiers Media S.A (Supplementary material).

COMPARATIVE AND METHODOLOGICAL PERSPECTIVES

Large-scale meta-analyses reveal striking parallels in gut microbiome dysbiosis among those with pediatric ALL, type 1 diabetes mellitus (T1DM), and allergic conditions, despite their distinct clinical presentations. ALL patients exhibit significantly reduced gut microbiome α-diversity at diagnosis compared to healthy controls, mirroring patterns observed for eczema and T1DM. Meta-analysis identifies the shared depletion of Lactobacillus (62% of ALL, 58% of T1DM, and 51% of eczema studies) and Bifidobacterium (54% of ALL, 49% of T1DM, and 44% of eczema studies) across disease states. Conversely, Enterococcus abundance is consistently elevated in ALL (73% of studies), T1DM (68%), and food allergy (61%) cohorts compared to controls. This taxonomic convergence extends to functional pathways, with butyrate synthesis genes (e.g., but and buk) being under-expressed in ALL gut microbiomes, while lipopolysaccharide biosynthesis pathways are enriched[12].

METHODOLOGICAL PITFALLS IN PEDIATRIC GUT MICROBIOME-LEUKEMIA RESEARCH

Investigating gut microbiome-leukemia interactions in children presents unique methodological challenges that require rigorous standardization to ensure robust findings. Researchers emphasize that variability in experimental design, sample processing, and bioinformatics pipelines can significantly distort microbial community profiles, particularly in vulnerable cohorts like pediatric leukemia patients. A critical pitfall lies in sample collection and storage protocols, as inconsistent fecal sampling methods (e.g., stool swab vs whole stool) or delays in freezing samples may alter microbial viability and DNA quality, skewing downstream sequencing results. Additionally, DNA extraction methods introduce substantial bias, with pediatric stool samples often containing a lower microbial biomass due to chemotherapy-induced dysbiosis, amplifying the risk of host DNA contamination and false negatives during the detection of taxa. The choice of sequencing platforms and bioinformatic tools further complicates cross-study comparisons; for example, different 16S rRNA gene regions (V4 vs V3–V4) and clustering algorithms (operational taxonomic unit vs amplicon sequence variant) yield divergent taxonomic resolutions, potentially obscuring leukemia-associated microbial signatures[13]. Inadequate control for confounders like antibiotic exposure, chemotherapy timing, and dietary changes – ubiquitous in pediatric oncology – can obscure true gut microbiome-leukemia associations. The longitudinal instability of microbial communities poses another pitfall, as frequent antibiotic courses and cytotoxic therapies induce rapid taxonomic shifts, complicating causal inferences about the role of baseline gut microbiomes in leukemogenesis[14]. Pediatric gut microbiome studies demand stringent controls for confounders, all of which disproportionately affect the microbial profiles of children compared to adults. The authors stress that the low microbial biomass in pediatric stool samples heightens contamination risks, requiring robust negative controls and DNA extraction methods that have been optimized for low-yield specimens. Future research must prioritize harmonized protocols across institutions to disentangle treatment effects from true disease-associated dysbiosis, enabling reproducible biomarker discovery for prognostic or therapeutic applications. Only through such rigorous adherence can pediatric gut microbiome-leukemia studies transcend observational associations and move toward insights[15].

GUT MICROBIOME BIOMARKERS FOR EARLY LEUKEMIA DETECTION

The gut microbiome has emerged as a promising frontier for identifying non-invasive biomarkers in oncology, with growing evidence implicating microbial dysbiosis in the pathogenesis and progression of hematologic malignancies. Recent studies reveal that specific microbial signatures and metabolite profiles in newly diagnosed acute myeloid leukemia (AML) patients exhibit predictive potential even before the manifestation of clinical symptoms. For instance, Enterococcus enrichment and the depletion of butyrate-producing taxa like Faecalibacterium and Roseburia are hallmarks of AML-associated dysbiosis, correlating strongly with SCFA levels and disease severity. Concurrently, randomization studies have established causal links between the gut microbiome composition and leukemia risk, identifying Blautia and Rikenellaceae RC9 as pro-leukemogenic taxa. Such findings position the gut microbiome as a dynamic biosensor of hematopoietic dysfunction, where early dysbiotic patterns may precede conventional diagnostic markers like cytogenetic abnormalities or blast cell proliferation. The integration of microbial biomarkers with metabolic profiling, particularly butyrate and acetate deficits, could enable risk stratification and interceptive strategies, offering a paradigm shift in leukemia screening[16]. A bidirectional randomization study leveraging genome-wide association data from 14306 individuals identified 10 gut microbial taxa with causal links to leukemia risk, offering mechanistic insights into microbial contributions to leukemogenesis. For acute ALL, Blautia and Lactococcus emerged as robust risk factors, while Slackia exhibited protective effects. In AML, the Rikenellaceae RC9 gut group, Anaerostipes, and the Lachnospiraceae ND3007 group were strongly pro-leukemogenic, whereas Acidaminococcaceae reduced AML risk. Chronic leukemia subtypes also displayed unique associations: Ruminococcaceae UCG011 and UCG014 elevated the risk of chronic myeloid leukemia, while Desulfovibrio protected against chronic lymphocytic leukemia. These taxa-specific signatures highlight the potential for using the gut microbiome to stratify leukemia risk before its clinical manifestation[17]. While these findings are promising, translating gut microbiome biomarkers into clinical practice requires addressing the heterogeneity of study designs, standardizing sampling protocols, and validating across diverse cohorts. Integrating metagenomic sequencing with profiling and machine learning models could enhance predictive accuracy, enabling risk stratification before overt hematologic abnormalities emerge. The gut microbiome exhibits significant potential as a dynamic biomarker for early leukemia detection, with microbial diversity loss and taxon-specific dysbiosis offering predictive insights into preleukemic states. However, clinical implementation hinges on overcoming methodological inconsistencies and validating causal relationships in longitudinal cohorts. Future research must prioritize harmonized protocols and mechanistic studies to unlock the full diagnostic utility of the gut microbiome in pediatric oncology[18].

INNOVATIVE GUT MICROBIOME-TARGETED THERAPIES IN PEDIATRIC ONCOLOGY

The gut microbiome has emerged as a critical determinant of clinical outcomes in pediatric oncology, fundamentally reshaping our understanding of treatment efficacy, toxicity, and long-term survivorship. As evidence continues to accumulate demonstrating the profound impact of microbial dysbiosis on treatment-related complications, including graft-versus-host disease (GvHD), BSIs, and therapeutic resistance, the development of innovative gut microbiome-targeted interventions has become an urgent clinical priority. Researchers have comprehensively demonstrated that nutritional strategies can serve as cost-effective and risk-appropriate methods for modulating the intestinal ecosystem to improve clinical outcomes in allogeneic HSC transplantation recipients. Their work highlights how pre-transplant diet composition affects microbial resilience during treatment, how the choice of enteral or parenteral nutrition fundamentally alters the gut ecosystem structure (Figure 2)[19], and how specific dietary components can be strategically employed to promote beneficial microbial populations while suppressing pathogenic overgrowth. The treatment of children presents unique considerations for gut microbiome-targeted therapies, as their gut microbiome undergoes continuous developmental changes while simultaneously interfacing with a maturing immune system. In comparison to adults, pediatric cancer patients exhibit greater microbial plasticity, potentially offering enhanced responsiveness to interventional strategies but also requiring age-specific safety considerations. The emerging therapeutic landscape encompasses diverse approaches, including targeted nutritional interventions, carefully selected probiotic supplementation, the prebiotic enhancement of beneficial microbial populations, innovative postbiotic administration, and pioneering applications of FMT in select cases. A growing body of evidence supports the clinical application of microbiota-targeted therapies in pediatric leukemia. The most clinically established microbiota-targeted interventions with demonstrated benefit – including probiotics, prebiotics, synbiotics, postbiotics, nutritional modulation, and FMT – are summarized in Table 1[8,19,20].

Figure 2 Lactose-driven enterococcus expansion in allogeneic hematopoietic stem cell transplantation recipients[19]. Dietary lactose that is unabsorbed due to conditioning-induced lactase loss accumulates in the gut lumen and serves as a substrate for Enterococcus species, leading to intestinal domination, enhanced mucosal barrier disruption, and an increased risk of acute graft-versus-host disease and bloodstream infections. Lactose avoidance or enzymatic supplementation restores community balance and reduces transplant-related complications. APC: Antigen presenting cell; CHT: Chemotherapy. Citation: Muratore E, Leardini D, Baccelli F, Venturelli F, Prete A, Masetti R. Nutritional modulation of the gut microbiome in allogeneic hematopoietic stem cell transplantation recipients. Front Nutr 2022; 9: 993668. Copyright ©The Author(s) 2022. Published by Frontiers Media S.A (Supplementary material).

Table 1 Microbiota-targeted interventions in pediatric leukemia.

Intervention	Agent	Clinical implications	Ref.
Probiotics	Lactobacillus rhamnosus GG	Reduced gastrointestinal toxicity; reduced antibiotic use	Martyniak et al[20], 2023
Probiotics	Bifidobacterium breve Yakult	Fewer fever episodes; reduced pathogens	Todor and Ichim[8], 2025
Prebiotics	Inulin	Increased chemo efficacy; reduced complications	Martyniak et al[20], 2023
Prebiotics	Pectin	Protects gut barrier during chemo	Martyniak et al[20], 2023
Postbiotics	Butyrate	Anti-inflammatory; anti-leukemic	Martyniak et al[20], 2023
Synbiotics	LactoCare (multi-strain + prebiotic)	96% reduced diarrhea	Martyniak et al[20], 2023
FMT	Fresh/frozen FMT	86% response in steroid-refractory GvHD	Todor and Ichim[8], 2025
Nutrition	Enteral nutrition	Better hematopoietic stem cell transplantation outcomes; reduced GvHD	Muratore et al[19], 2022

FMT: Fecal microbiome transplantation; GvHD: Graft-versus-host disease.

Current evidence suggests that gut microbiome-targeted therapies can significantly reduce treatment-associated toxicities, including mucositis, complications from infections, and severe GvHD gastrointestinal (GI) manifestations, while potentially enhancing immune reconstitution and overall treatment tolerance[19]. The therapeutic potential of probiotics in pediatric leukemias has been demonstrated through several clinical trials examining their impact on treatment-related complications and immune recovery. The Bifidobacterium breve strain Yakult has shown promise in preventing infections among pediatric leukemia patients, with clinical trials demonstrating fewer episodes of fever, reduced antibiotic utilization, and decreased levels of pathogenic Enterobacteriaceae in fecal samples[8,20]. L. rhamnosus GG represents one of the most extensively studied probiotic strains in pediatric oncology, with a randomized controlled trial demonstrating significant reductions in GI toxicity among children receiving chemotherapy for ALL. Martyniak et al[20] discovered that only 30% of patients who received probiotic supplementation experienced GI symptoms compared to 63% in the control group, with nearly two-fold reductions in antibiotic requirements, hospitalization needs, and septicemia episodes. Additionally, probiotic supplementation with Lactobacilli species has been associated with improved tolerance for antimicrobial therapy, with significant reductions in treatment-related nausea and vomiting, leading to better medication acceptance and adherence. Beyond currently established strategies, several innovative and experimental microbiome-based interventions are under early clinical evaluation. These emerging approaches – including biomarker-guided microbiome monitoring, autologous FMT, and immunomodulatory agents – are outlined in Table 2[4,8-10,19,20].

Table 2 Emerging interventions in pediatric leukemia.

Intervention	Agent	Clinical implications	Ref.
Lactoferrin	Bovine lactoferrin 200 mg/day	Prevents dysbiosis; reduced febrile neutropenia	Martyniak et al[20], 2023
Melatonin	Melatonin (various)	Increased microbiome diversity	Martyniak et al[20], 2023
Biomarkers	Quantitative polymerase chain reaction (Ruminococcaceae/Veillonella)	91% specificity for neutropenia risk	Sørum et al[9], 2025
VOC panel	Fecal VOC profiling	Predicts bloodstream infection 15 days pre-symptoms	van de Velde et al[4], 2022
Autologous fecal microbiome transplantation	Patient’s own stool	Personalized microbiome recovery	Todor and Ichim[8], 2025
Lactose-free diet	Conditioning/hematopoietic stem cell transplantation	Prevents Enterococcus blooms; reduced graft-versus-host disease	Muratore et al[19], 2022
High-fiber diet	≥ 5 g fiber/day	27% reduced leukemia risk (epidemiological)	Kameri et al[10], 2024

VOC: Volatile organic compound.

FMT IN PEDIATRIC ONCOLOGY

FMT represents the most comprehensive gut microbiome restoration strategy, offering the rapid reconstitution of microbial diversity and functional capacity in severely dysbiotic patients. In pediatric leukemia patients, FMT has shown particular promise for treating steroid-refractory GvHD and multidrug-resistant bacterial colonization. Clinical series examining FMT in pediatric patients with GI GvHD have demonstrated response rates of approximately 86%, with clinical improvement occurring within 4 days compared to 48 days for control groups. Successful FMT interventions resulted in the restoration of beneficial microbial populations, including Bacteroides fragilis and F. prausnitzii, along with significant increases in the overall bacterial biomass and diversity. Complete response rates 30 days, 60 days, and 90 days post-FMT were 42%, 74%, and 84%, respectively, representing substantial improvements over conventional immunosuppressive therapy alone. For multidrug-resistant bacterial decolonization in pediatric ALL patients, FMT achieved an 80% decolonization rate within one week of administration, though recurrent colonization occurred in the majority of patients, suggesting the need for repeat procedures or combination approaches. Safety profiles from immunocompromised children have shown predominantly mild adverse events, including transient nausea and abdominal pain in approximately 86% of patients[19].

PREBIOTIC AND POSTBIOTIC STRATEGIES FOR GUT MICROBIOME RESTORATION

Prebiotic interventions offer a safer alternative to live microbial supplementation by selectively promoting the growth of beneficial indigenous bacteria while avoiding the risks associated with exogenous microbial administration. Inulin, a fructan-type prebiotic naturally found in bananas, onions, garlic, and artichokes, has demonstrated synergistic effects with conventional chemotherapy agents. Preclinical studies have shown that inulin supplementation enhances the cytotoxic properties of doxorubicin, potentially allowing for dose reduction while maintaining therapeutic efficacy. The mechanisms underlying inulin's beneficial effects include the stimulation of butyrate and propionate production by indigenous Bifidobacterium and Lactobacillus species, contributing to enhanced intestinal barrier function and reduced systemic inflammation. In murine leukemia models, inulin supplementation significantly increased cecal concentrations of SCFAs and reduced hepatic metastases, suggesting both local and systemic anti-tumor effects. Pectin, a water-soluble fiber abundant in citrus fruits, apples, and legumes, has shown protective effects against chemotherapy-induced intestinal damage. In experimental models administering methotrexate, pectin supplementation significantly reduced intestinal permeability, improved mucosal integrity, and decreased bacterial translocation into systemic circulation. Pectin oligosaccharides have additionally demonstrated efficacy in reducing cancer-associated cachexia, with studies showing decreased anorexia and the preservation of adipose tissue mass in leukemia models. Postbiotic interventions represent an emerging therapeutic approach, harnessing the beneficial effects of microbial metabolites without the safety concerns associated with live microorganisms. SCFAs, particularly butyrate, acetate, and propionate, serve as key postbiotic mediators with established anti-inflammatory and anti-neoplastic properties. Butyrate exhibits dose-dependent effects on leukemic cell viability, with high concentrations (> 1.5 mmol/L) inducing apoptosis through caspase-3 activation and reducing cell viability by up to 60% in acute leukemia cell lines. Additionally, butyrate supplementation significantly decreases pro-inflammatory chemokine production, including C-C motif chemokine ligand 2 (CCL2) and CCL5, which may reduce metastatic potential and modulate the tumor microenvironment. The anti-cancer mechanisms of butyrate involve histone deacetylase inhibition, which normalizes the epigenetic dysregulation that is characteristic of hematologic malignancies. Clinical evidence supporting postbiotic interventions comes from studies examining enteral vs parenteral nutrition in pediatric hematopoietic stem cell transplantation recipients, where enteral feeding promoted the faster recovery of SCFA production and the restoration of a beneficial gut microbiome. Table 3 presents a structured, phase-specific framework for tailoring microbiome interventions to align with these evolving clinical needs throughout pediatric leukemia care[8,10,11,19,20]. Meta-analyses have demonstrated that enteral nutrition reduces the incidence of acute GvHD, in particular severe grades III-IV and GI manifestations, potentially through improved gut eubiosis and enhanced SCFA-mediated immune modulation[20].

Table 3 Phase-specific interventions in pediatric leukemia.

Phase	Key intervention	Agent	Clinical note	Ref.
Pre-diagnosis	Maternal probiotics	L. rhamnosus GG + Bifidobacterium infantis	38% reduced offspring ALL risk	Kameri et al[10], 2024
Pre-diagnosis	Breastfeeding	Exclusive > 6 months	27% reduced ALL risk	Kameri et al[10], 2024
Chemo	Probiotics	L. rhamnosus GG	Reduced gastrointestinal toxicity/infections	Martyniak et al[20], 2023
Chemo	Prebiotics	Inulin + butyrate	Increased tolerance	Martyniak et al[20], 2023
Chemo	Antibiotic stewardship	Reduced fluoroquinolone use	Preserves commensals	Dunn et al[11], 2022
HSCT	Enteral nutrition	Enteral nutrition vs parenteral nutrition	Maintains gut structure	Muratore et al[19], 2022
HSCT	Lactose avoidance	Lactose-free diet	Reduced Enterococcus dominance	Muratore et al[19], 2022
Recovery	Microbiome restoration	Fecal microbiome transplantation/targeted probiotics	Reduced late effects 29%	Todor and Ichim[8], 2025

ALL: Acute lymphoblastic leukemia; L. rhamnosus: Lactobacillus rhamnosus; HSCT: Hematopoietic stem cell transplantation.

NOVEL CONCEPTUAL FRAMEWORKS

The rapidly evolving landscape of gut microbiome-targeted therapeutics in pediatric leukemia necessitates the development of robust regulatory frameworks and comprehensive safety protocols that can adequately address the unique challenges posed by these innovative interventions. The emergence of gut microbiome-based therapies has fundamentally challenged traditional regulatory paradigms, requiring substantial adaptations of European and global regulatory frameworks to effectively accommodate these novel therapeutic modalities. The regulatory landscape has evolved to recognize distinct categories of gut microbiome interventions, including live biotherapeutic products (LBPs), phage therapy-based medicinal products, and gut microbiome transplantation preparations, each requiring tailored oversight approaches. The European Pharmacopoeia has established a specific definition of LBPs as "medicinal products containing live microorganisms (bacterial or yeasts) for human use", primarily addressing quality requirements for orally or vaginally administered products. However, the development of novel delivery routes, including topical administration and systemic injection, continues to create new regulatory challenges regarding manufacturing process controls and safety assessment protocols. The implementation of the European Union’s new Regulation on Substances of Human Origin (SoHO) represents a pivotal advance in harmonizing the regulatory status of FMT preparations across member states. This regulation establishes comprehensive requirements for organizations involved in gut microbiome-related activities, mandating registration as a "SoHO entity" and authorization as a "SoHO establishment" for specific activities, including donor registration, collection, processing, quality control, storage, and distribution. This regulatory evolution emphasizes the importance of using science-driven benefit-risk analysis to demonstrate positive therapeutic ratios within specific pediatric leukemia patients, while establishing standardized protocols for donor screening, product characterization, and quality assurance[21]. The integration of gut microbiome science into pediatric oncology requires sophisticated conceptual frameworks that address the unique developmental, immunological, and therapeutic challenges inherent to children. The pediatric gut microbiome exhibits fundamental differences from its adult counterpart, with rapid compositional changes occurring throughout infancy and childhood, necessitating age-stratified research approaches and interventional strategies. Key questions have emerged regarding the optimal methodological approaches for studying gut microbiome-leukemia interactions, including determining which classes of antibiotics and clinical factors most significantly lead to intestinal domination, identifying crucial gut microbiome components that provide resistance against pathogenic species, and establishing whether environmental controls can minimize treatment-induced dysbiosis. Conceptual frameworks must encompass the complex interplay between gut microbiome modulation and immune system function, with particular emphasis on how gut microbial communities influence responses to immunotherapies, cellular therapies, and anti-tumor vaccines in pediatric patients. Critical considerations include whether the pediatric gut microbiome differs sufficiently from that of adults to affect the treatment efficacy for tumors with similar histology or genetic drivers, and which specific biomarkers best predict therapeutic outcomes. Frameworks should encompass a longitudinal assessment of gut microbiome-chemotherapy interactions, examining which therapeutic agents are most affected by microbial metabolism and whether local contact within the tumor microenvironment is necessary for metabolism-mediated dysbiosis to affect treatment efficacy. Long-term considerations within these conceptual frameworks should also address whether gut microbiome dysbiosis persists for years after cancer treatment and whether these persistent changes are associated with specific late effects, such as insulin resistance or cardiomyopathy. Frameworks must also emphasize the need for prospective studies examining gut microbiome restoration strategies, including the optimal timings for interventions, the selection of appropriate probiotic strains or prebiotic supplements, and the development of personalized approaches based on individual patient gut microbiome profiles. Safety considerations should be integrated throughout, recognizing that immunocompromised pediatric patients require specialized protocols that balance therapeutic potential with infection risks[22].

SAFETY CONSIDERATIONS AND RISK ASSESSMENT PROTOCOLS

The implementation of gut microbiome-targeted therapies in immunocompromised pediatric leukemia patients necessitates comprehensive safety protocols that acknowledge the unique vulnerabilities of this population to treatment-related complications. Clinical evidence from cross-sectional studies demonstrates that hospitalized immunocompromised children, including transplantation recipients and those with cancer and sickle cell disease, exhibit distinct microbial profiles compared to immunocompetent cohorts, though alpha diversity measures may not differ significantly between groups. However, lower microbial diversity was independently associated with the use of proton pump inhibitors or antibiotics, including prophylactic penicillin in patients with sickle cell disease, identifying specific medication exposures as critical risk factors for dysbiosis in immunocompromised pediatric patients. Safety profiles reveal that immunocompromised children experience heightened susceptibility to medication-induced microbial perturbations, necessitating specialized monitoring approaches that account for the complex interplay between underlying immune dysfunction and therapeutic interventions (Figure 3)[23]. Risk assessment protocols must incorporate a comprehensive evaluation of patient-specific factors, including underlying immunodeficiency etiology, concurrent medications, prior antibiotic exposure history, and baseline gut barrier integrity, while accounting for the heterogeneity of immunocompromising conditions in pediatric patients. Inborn errors of immunity present particularly complex challenges, as patients with conditions such as common variable immunodeficiency, Wiskott-Aldrich syndrome, severe combined immunodeficiency, and chronic granulomatous disease demonstrate characteristic patterns of microbial dysbiosis that require specialized therapeutic approaches. The consequences of an increase in pro-inflammatory bacteria or a reduction in anti-inflammatory bacteria in these patients create distinct GI, respiratory, and cutaneous symptoms and signs linked to dysbiosis, emphasizing the importance of gut microbiome identification and targeted intervention strategies. Functional and compositional differences in gut microbiomes among patients with inborn errors of immunity necessitate personalized risk assessment approaches that consider the specific genetic abnormalities disrupting a normal host-immune response or immune regulation[24]. Safety monitoring protocols require the establishment of standardized adverse event reporting systems specifically designed for pediatric patients receiving gut microbiome interventions, with particular attention paid to age-related vulnerability factors. Clinical experience with FMT in Chinese children demonstrates that while short-term adverse events occurred for approximately 26% of procedures, the majority were self-limiting and resolved within 48 hours. However, an age greater than 72 months appeared to be correlated with more adverse events compared to younger children, and immune state emerged as an independent risk factor for adverse event occurrence, with immunodeficient patients having a risk ratio of 3.105 for developing complications. The most common short-term adverse events included abdominal pain, diarrhea, fever, and vomiting, though severe adverse events and mortality remained rare, occurring in approximately 4% and 2% of cases, respectively[25]. The establishment of comprehensive safety frameworks must address the documented risks of invasive infections associated with gut microbiome interventions, particularly probiotic supplementation in vulnerable pediatric patients. Systematic analysis of invasive infections related to probiotic use in children reveals that sepsis represents the most observed condition, affecting approximately 69% of documented cases, with bacteremia or fungemia occurring in an additional 29% of patients. The causative organisms include Lactobacillus species (35%), Saccharomyces species (29%), Bifidobacterium species (31%), Bacillus clausii (4%), and Escherichia coli (2%), with most affected patients being younger than two years old and having at least one condition facilitating the development of invasive infections. Critical risk factors include prematurity (55%) and intravenous catheter use (51%), highlighting the heightened vulnerability of hospitalized pediatric patients to probiotic-associated systemic infections[26]. Future safety frameworks must prioritize the development of refined risk stratification tools that can identify the pediatric patients most likely to benefit from gut microbiome interventions while minimizing the exposure risks for vulnerable populations. The establishment of standardized protocols for donor screening, product characterization, and post-treatment surveillance systems that have been specifically designed for pediatric immunocompromised patients represents an urgent priority for clinical implementation. Enhanced safety monitoring should incorporate real-time gut microbiome assessment capabilities to distinguish treatment-related complications from underlying disease progression, particularly given the complex medical histories and multiple concurrent therapies that are typical in children with leukemia. The development of age-appropriate formulations and delivery systems that minimize contamination risks while maintaining therapeutic efficacy remains critical need relating to the current development of gut microbiome therapeutics[27].

Figure 3 Impact of antibiotics, proton pump inhibitors, and Clostridium difficile colonization on gut microbial diversity in hospitalized children[23]. Heatmaps of α-diversity metrics comparing species richness (left) and Shannon index (right) across the entire cohort. Each point represents an individual patient; boxplots denote the median, interquartile range, and full range. IC: Immunocompromised. Citation: Mohandas S, Soma VL, Tran TDB, Sodergren E, Ambooken T, Goldman DL, Weinstock G, Herold BC. Differences in gut microbiome in hospitalized immunocompetent vs. immunocompromised children, including those with sickle cell disease. Front Pediatr 2020; 8: 583446. Copyright ©The Author(s) 2020. Published by Frontiers Media S.A (Supplementary material).

DISCUSSION

The emerging evidence regarding gut microbiome dysbiosis in pediatric leukemia represents a paradigm shift for understanding hematologic malignancy pathogenesis, extending beyond traditional genetic and environmental risk factors to encompass the complex gut microbiome-immune axis that governs childhood cancer development and treatment outcomes. Recent investigations have demonstrated that gut microbiome dysbiosis constitutes a fundamental component of hematologic malignancy progression, with distinctive microbial signatures observed not only in pediatric ALL but across a broader spectrum of childhood hematologic cancers, including non-Hodgkin lymphoma, AML, and Hodgkin lymphoma. The comprehensive involvement of the gut microbiome suggests the existence of shared pathophysiologic mechanisms underlying diverse hematologic malignancies, wherein microbial community disruption compromises immune surveillance, promotes chronic inflammation, and facilitates malignant transformation through interconnected pathways that transcend specific cancer subtypes. The demonstration that adolescent Hodgkin lymphoma survivors exhibit reduced gut microbial diversity compared to unaffected co-twin controls provides compelling evidence that gut microbiome dysbiosis persists beyond active disease phases and may represent both a causative factor and a long-term consequence of hematologic malignancy development. Furthermore, these findings extend beyond leukemia to encompass solid pediatric tumors, where distinctive gut microbiome profiles characterized by increased Enterococcus abundance and reduced beneficial taxa have been identified, suggesting that gut microbiome dysbiosis represents a unifying feature of pediatric oncogenesis that warrants comprehensive investigation across all childhood cancer types[10]. The mechanistic foundations underlying gut microbiome-immune interactions in pediatric hematologic malignancies involve sophisticated regulatory networks that modulate both innate and adaptive immune responses through bacterial metabolite production, direct cellular signaling, and epigenetic modifications that collectively influence leukemogenesis and treatment efficacy. The fundamental mechanisms underlying gut-immune axis interactions in hematologic malignancies share common pathways across different blood disorders, as demonstrated by recent comprehensive analyses of autoimmune hematologic conditions. The gut microbiome influences immune homeostasis through multiple interconnected mechanisms, including the production of SCFAs such as butyrate, propionate, and acetate, which enhance regulatory T cell (Treg) differentiation and suppress inflammatory cytokines including interleukin-6, tumor necrosis factor-alpha, and interferon-gamma, thereby promoting anti-inflammatory responses both systemically and within the gut microenvironment. Pattern recognition receptor (PRR) signaling pathways, particularly through TLRs and nucleotide-binding oligomerization domain-like receptors, play crucial roles in distinguishing commensal bacteria from pathogenic microorganisms, normally regulating immune tolerance by limiting unnecessary immune activation. However, under conditions of gut dysbiosis, aberrant PRR signaling may trigger chronic immune activation, disrupting the delicate balance between Treg cells and effector T cells (Th1/Th17), thereby promoting a pro-inflammatory milieu that could contribute to the pathogenesis of pediatric leukemia through compromised immune surveillance and enhanced inflammatory responses. Furthermore, the gut microbiome maintains intestinal barrier integrity through tight junction proteins (e.g., occludin, claudin, and zonulin) that regulate intestinal permeability, preventing the translocation of bacterial endotoxins such as LPS into systemic circulation; the dysbiosis-induced compromising of this gut barrier can elevate circulating LPS levels, trigger systemic inflammation, and aberrantly activate monocytes and dendritic cells, factors that may contribute to immune dysregulation and create a favorable environment for leukemogenesis in susceptible pediatric populations[28]. The observation that specific bacterial taxa such as A. muciniphila and F. prausnitzii demonstrate protective effects against the progression of hematologic malignancies through enhanced immune checkpoint modulation and improved therapeutic responses highlights the potential for targeted gut microbiome interventions to augment conventional cancer therapies[29]. The identification of steroid hormone metabolites as potential mediators between immune cell populations and lymphoid leukemia development further emphasizes the existence of complex biochemical networks[30]. Clinical trials investigating probiotic administration in pediatric oncology patients have demonstrated significant reductions in treatment-related GI toxicity[5]. The successful application of FMT in pediatric patients with treatment-refractory GvHD has achieved response rates exceeding 80%[31]. Advanced gut microbiome-targeted approaches include precision probiotic selection based on the microbial profiles of individual patients and the integration of artificial intelligence and machine learning algorithms for predicting optimal gut microbiome interventions[32]. Evidence demonstrates that baseline gut microbiome diversity serves as an independent predictor of treatment-related complications[18]. Also, it has been observed that specific microbial signatures can predict treatment responses and long-term outcomes in pediatric ALL[10]. The interplay between the gut microbiome and pediatric leukemia is increasingly recognized as both a driver of disease pathogenesis and a modifiable factor influencing treatment outcomes. As research continues to unravel the complexity of gut microbiome-immune interactions, there is growing optimism that targeted modulation of the gut microbiome could offer novel strategies for prevention, risk stratification, and supportive care in the area of pediatric hematologic malignancies. Future studies should focus on large-scale, multicenter cohorts and standardized methodologies to validate gut microbiome-based biomarkers and interventions.

CONCLUSION

The gut microbiome has emerged as a pivotal determinant of pediatric leukemia pathogenesis, prognosis, and therapeutic outcomes, fundamentally reshaping our understanding of childhood hematologic malignancies and opening up unprecedented avenues for the application of precision medicine. This comprehensive review illuminated the complex bidirectional relationship between microbial dysbiosis and leukemogenesis, demonstrating how early-life gut microbiome immaturity may predispose genetically susceptible children to ALL through disrupted immune surveillance and chronic inflammatory pathways. The clinical implications of gut microbiome research in pediatric leukemia extend far beyond diagnostic applications, encompassing predictive biomarkers relating to treatment complications, personalized therapeutic strategies, and long-term survivorship considerations. Furthermore, the development of innovative gut microbiome-targeted therapies, including probiotics, prebiotics, postbiotics, and FMT, offers promising strategies to mitigate treatment-related toxicities while potentially enhancing therapeutic efficacy through immune modulation and metabolic reprogramming. However, the translation of gut microbiome research into routine clinical practice faces significant methodological, regulatory, and safety challenges that must be systematically addressed to realize the full therapeutic potential of these interventions. Future research priorities must encompass mechanistic studies to elucidate causal relationships between specific microbial taxa and leukemogenesis, longitudinal cohort studies to validate predictive biomarkers, and randomized controlled trials to demonstrate the safety and efficacy of gut microbiome-targeted interventions. As we advance toward an era of gut microbiome-informed precision medicine, the integration of these insights into routine pediatric oncology practice holds the potential to transform the landscape of pediatric leukemia care.