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 range of malignant and non-malignant hematologic disorders in both pediatric and adult patients. Despite significant therapeutic advances, these procedures remain associated with substantial morbidity and mortality due to complications such as acute and chronic graft-versus-host disease (GvHD), severe infections, relapse, delayed immune reconstitution, and therapy-related toxicities. Increasing evidence indicates that the gut microbiota (GM) plays a critical role in modulating immune responses and influencing clinical outcomes after HCT. Reduced microbial diversity and domination by pathogenic taxa have been associated with higher rates of GvHD, bloodstream infections, transplant-related mortality, and inferior overall survival in adult populations. Conversely, the presence of beneficial commensal bacteria and microbial metabolites, such as short-chain fatty acids, appears to support epithelial barrier integrity and immune regulation. Emerging data also suggest that microbiota composition may influence the efficacy and toxicity profile of CAR-T therapy. However, most available evidence derives from adult cohorts, and data in pediatric patients remain limited. Children differ from adults in terms of microbiota development, immune maturation, environmental exposures, and treatment history. As a result, microbiota-host interactions in pediatric transplant recipients may follow distinct patterns, and extrapolation from adult studies may be inadequate. Moreover, the mechanisms linking microbiota alterations to transplant-related complications are not fully understood in either population. Antibiotic exposure represents a major determinant of microbiota disruption in the peri-transplant period. Broad-spectrum antibiotics are frequently administered for prophylaxis or treatment of infections, but their use can significantly reduce microbial diversity, promote dysbiosis, and facilitate colonization by multidrug-resistant organisms. These changes may negatively affect immune homeostasis and clinical outcomes. Antimicrobial resistance is an additional concern in immunocompromised patients, emphasizing the importance of antibiotic stewardship strategies. However, the biological impact of antibiotic policies on microbiota composition and transplant outcomes has not been comprehensively characterized, particularly in pediatric settings. This study is designed as a prospective, observational, multi-omic investigation of pediatric and adult patients undergoing allogeneic HCT and advanced cellular therapies. The project aims to integrate longitudinal clinical data with comprehensive molecular profiling of both the gut microbiota and the host immune system. Biological samples, including stool and peripheral blood, will be collected at predefined time points before and after transplantation or cellular therapy. Gut microbiota composition and diversity will be analyzed using sequencing-based approaches, while microbial functional potential will be explored through metagenomic and metabolomic analyses. Host responses will be characterized through transcriptomic profiling, immune cell phenotyping, cytokine assessment, and evaluation of immune reconstitution dynamics. The primary objective is to identify microbiota- and host-derived biomarkers associated with key clinical outcomes, including acute and chronic GvHD, severe infections, antimicrobial resistance patterns, relapse, non-relapse mortality, overall survival, and therapy-related toxicities such as cytokine release syndrome in CAR-T recipients. Secondary objectives include assessing the impact of antibiotic exposure and stewardship strategies on microbiota diversity and function, comparing microbiota trajectories between pediatric and adult patients, and exploring associations between microbiota features and therapeutic response. Advanced bioinformatic and statistical methods will be used to integrate multi-omic datasets with detailed clinical variables, enabling identification of predictive signatures and mechanistic pathways. Age-stratified analyses will be performed to highlight pediatric-specific versus adult-specific patterns of microbiota-immune interaction. By providing a comprehensive and longitudinal characterization of the intestinal ecosystem and host immune responses in transplant and cellular therapy recipients, this study aims to improve understanding of the biological determinants of treatment outcomes. The results may contribute to the development of microbiota-informed risk stratification tools and support future personalized strategies, including optimized antibiotic stewardship and targeted microbiota-modulating interventions, ultimately improving survival and quality of life for patients.