Multi-omic Characterization of Pediatric and Adult Patients Undergoing Hematopoietic Stem Cell Transplantation and Advanced Cell Therapies

Allogeneic hematopoietic stem cell transplantation (HCT) and advanced cellular therapies, including chimeric antigen receptor T-cell (CAR-T) therapy, are potentially curative treatments for a wide spectrum of malignant and non-malignant hematologic diseases in both pediatric and adult patients. Indications include acute and chronic leukemias, lymphomas, myelodysplastic syndromes, bone marrow failure syndromes, primary immunodeficiencies, and selected inherited disorders. Despite major advances in conditioning regimens, donor selection, graft engineering, and supportive care, these interventions remain associated with significant morbidity and mortality. Acute and chronic graft-versus-host disease (GvHD), severe infectious complications, delayed immune reconstitution, relapse, organ toxicities, and treatment-related adverse events continue to represent major clinical challenges. In the CAR-T setting, additional toxicities such as cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), prolonged cytopenias, and infections further complicate patient management. Increasing evidence indicates that the gut microbiota plays a central role in modulating immune homeostasis and systemic inflammatory responses in the transplant setting. The intestinal microbiota constitutes a complex and dynamic ecosystem that interacts with the host through metabolic, immunologic, and epithelial pathways. A diverse and balanced microbial community supports intestinal barrier integrity, produces immunomodulatory metabolites such as short-chain fatty acids, and contributes to the regulation of innate and adaptive immune responses. Conversely, disruption of microbial diversity and overgrowth of opportunistic or pathogenic taxa, a condition commonly referred to as dysbiosis, has been associated with epithelial injury, systemic inflammation, and immune dysregulation. Observational studies in adult allogeneic HCT recipients have demonstrated that reduced microbiota diversity and domination by specific bacterial taxa correlate with increased risk of acute GvHD, bloodstream infections, transplant-related mortality, and inferior overall survival. Certain commensal bacteria appear to exert protective effects, potentially through promotion of regulatory T-cell differentiation and maintenance of mucosal barrier function. However, most available data derive from adult cohorts, and pediatric-specific evidence remains limited. Children differ substantially from adults with respect to microbiota development, immune maturation, environmental exposures, nutritional factors, and prior treatments. The intestinal microbiome evolves dynamically across childhood and adolescence, suggesting that microbiota-host interactions in pediatric transplant recipients may follow distinct patterns. Extrapolation from adult data may therefore be inadequate. Emerging research also suggests that the gut microbiota may influence the efficacy and toxicity of CAR-T therapy. Baseline microbiota composition and antibiotic exposure prior to lymphodepletion have been associated with differential CAR-T expansion, response rates, and incidence of inflammatory toxicities. Nonetheless, mechanistic insights remain incomplete, and the interplay between microbial ecology, host immune activation, and treatment outcomes requires comprehensive investigation in both adult and pediatric populations. Antibiotic exposure represents one of the most significant drivers of microbiota disruption during the peri-transplant period. Broad-spectrum antibiotics are frequently administered for prophylaxis or treatment of suspected or documented infections, particularly during neutropenia. While essential for infection control, these agents can markedly reduce microbial diversity, eliminate beneficial commensal organisms, and promote the expansion of resistant pathogens. The emergence of multidrug-resistant organisms poses an additional threat to immunocompromised patients. Several studies have suggested that certain antibiotic classes and cumulative exposure are associated with increased risk of GvHD and mortality, highlighting the importance of antibiotic stewardship. However, the biological impact of antimicrobial prescribing patterns on microbiota composition, antimicrobial resistance genes, and transplant outcomes remains insufficiently characterized, especially in pediatric settings. This study is designed as a prospective, observational, longitudinal investigation integrating multi-omic analyses in pediatric and adult patients undergoing allogeneic HCT or advanced cellular therapies, including CAR-T. The study does not alter standard therapeutic approaches; all treatments, conditioning regimens, graft sources, CAR-T products, and supportive care strategies are administered according to institutional standards and clinical guidelines. The objective is to comprehensively characterize host-microbiota interactions and identify biological signatures associated with clinically relevant outcomes. Participants are enrolled prior to conditioning or lymphodepletion and followed longitudinally through early and late post-treatment phases. Clinical data are systematically collected, including demographic characteristics, underlying diagnosis, prior therapies, conditioning intensity, donor type, antimicrobial exposure, infection episodes, GvHD occurrence and severity, relapse, immune reconstitution parameters, CAR-T-related toxicities, and survival outcomes. Biological samples are obtained at predefined time points, including baseline and multiple post-treatment intervals, as well as at the onset of clinically significant events when feasible. Stool samples are collected to assess gut microbiota composition, diversity, and functional potential using sequencing-based approaches such as 16S rRNA gene sequencing and shotgun metagenomics. These analyses allow characterization of taxonomic profiles, microbial gene content, and antimicrobial resistance determinants. Peripheral blood samples are obtained to evaluate immune cell subsets, cytokine profiles, transcriptomic signatures, and circulating metabolites. Metabolomic analyses explore both host- and microbiota-derived metabolites that may influence immune regulation and epithelial integrity. The primary aim of the study is to identify microbiota- and host-derived biomarkers associated with key outcomes after HCT and CAR-T therapy, including GvHD, severe infections, colonization or infection with multidrug-resistant organisms, relapse, non-relapse mortality, overall survival, and treatment-related toxicities such as CRS and ICANS. Secondary objectives include characterization of longitudinal microbiota dynamics, evaluation of the impact of antibiotic exposure on microbial diversity and resistome profiles, comparison of pediatric and adult microbiota trajectories, and exploration of associations between microbiota features and immune reconstitution or therapeutic response. Advanced bioinformatic and statistical methods are employed to integrate microbiome, transcriptomic, metabolomic, and immunophenotypic data with detailed clinical variables. Multivariate modeling and machine learning approaches may be applied to identify predictive signatures and develop risk stratification models. Age-stratified analyses allow identification of pediatric-specific and adult-specific patterns of host-microbiota interaction. The study is conducted in accordance with ethical principles and applicable regulatory requirements. Written informed consent is obtained from adult participants and from parents or legal guardians of pediatric participants, with assent from minors when appropriate. Biological sampling is coordinated with routine clinical procedures whenever possible to minimize additional burden. All data are de-identified to ensure confidentiality.

By integrating comprehensive molecular profiling with longitudinal clinical follow-up, this study aims to generate an in-depth understanding of the biological determinants of outcomes in transplant and cellular therapy recipients. The identification of robust biomarkers and mechanistic pathways may support the development of personalized, microbiota-informed strategies, including optimized antimicrobial stewardship and future microbiota-targeted interventions, ultimately improving survival and reducing treatment-related complications in both pediatric and adult populations.