The immunological composition of the microenvironment has shown relevance for diagnosis, prognosis, and therapy in solid tumors but remains underexplored in acute leukemias. We investigated the significance of the acute myeloid leukemia (AML) bone marrow microenvironment in predicting chemosensitivity and long-term remission in pediatric patients. We analyzed 32 non-promyelocytic pediatric AML patients at diagnosis using a NanoString PanCancer IO 360 assay, RNA sequencing, and deep-phenotype flow cytometry analyses. The findings were validated using the pediatric TARGET AML dataset. A short signature of three interferon (IFN)-related genes (GBP1, PARP12, and TRAT1) distinguished patients with chemosensitive disease and reduced minimal residual disease after induction chemotherapy. The signature stratified patients overall, and within the clinically defined “standard-risk” group, patients with high gene expression at diagnosis had significantly longer overall survival. The leukemia microenvironment associated with this signature showed enrichment of non-exhausted CD4⁺ and CD8⁺ T cytotoxic lymphocytes and expansion of CD8⁺ T effector memory cells re-expressing CD45RA (TEMRA) in patients with a favorable prognosis. Our results show the importance of the bone marrow microenvironment in pediatric AML and provide tools for a refined stratification of “standard-risk” patients, lacking adequate risk-oriented therapies. They also offer a promising guide for tackling immune pathways and exploiting immune-targeted therapies.

Background: Graft versus host disease (GVHD) and the graft versus tumor (GVT) effect after allogeneic hematopoietic cell transplantation (allo-HCT) result from complex interactions between the donor immune system and the recipient environment. High-temporal longitudinal monitoring might be necessary to identify triggering events of GVHD and GVT and to intercept these events before their occurrence. But it would require an overall considerable amount of blood by venipuncture, which is unfeasible in such a fragile population. Methods: In this study, we implemented a targeted multiplex microfluidics q-PCR-based transcriptional fingerprint assay (TFA) on 50 µL of blood collected by a simple fingerstick to evaluate post-allo-HCT systemic immune perturbations associated with the development of GVHD. Fluctuations of a panel of 264 genes were measured in 31 allo-HCT patients by frequent (weekly or biweekly) analysis of 50 µL serial blood samples. Cross-sectional and longitudinal analyses correlated with detailed clinical annotations were performed. Results: Signatures of neutrophil activation and interferon (IFN) characterized the onset of acute GVHD, while an ongoing cytotoxic response was modulated in chronic mild GVHD and protein-synthesis and B-cell-related signatures characterized late acute/overlap GVHD. An unexpected erythroid signature distinguished patients with acute and mild chronic GVHD. Conclusions: Our micro-invasive approach unveiled the molecular heterogeneity of GVHD and identified hierarchically important biological processes conducive to different forms of GVHD. These findings increase our understanding of GVHD and reveal potentially targetable alterations. This approach might be implemented clinically to intercept GVHD before its occurrence and to modulate therapeutic interventions accordingly.