Single-Cell Transcriptomic Analyses Reveal Distinctive Immune Features between Childhood and Adult Acute Myeloid Leukemia

Background and Aims

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy with age-specific differences in genomics and drug responses being previously identified. This study aimed to further capture the transcriptomic differences of malignant and immune compartments between pediatric and adult AML and gain insights into their unique biology.

Method

ScRNA-seq data from 65 pediatric and 35 adult AML samples and 19 healthy donors (HD) were retrieved from six published and one in-house datasets. Unified diagnostic data were subjected to pre-processing prior to sample integration, cell type classification, and cell trajectory analysis. Differentially expressed genes (DEGs) between children and adult and functional enrichment in various cell types were determined by DESeq2 and ClusterProfiler.

Results

In the myeloblast compartment, 20 cell types were identified and cell diversity (Shannon & Simpson indexes) was significantly higher in pediatric AML(p<0.05). By ward.D2 clustering, majority of samples were clustered into two categories: monocyte-like (Mono-like) and hematopoietic stem and progenitor cells (HSPC-like) clusters. Significantly, more HSPC-like enriched blasts and fewer Mono-like blasts were observed in the pediatric AML (p<0.01). Specific genetic subtypes of AML were found to be associated with different clusters and distinct between pediatric and adult AML. In children, samples with CBFB-MYH11 were enriched in Mono-like (p<0.05). In adults, DNMT3A^mut samples were predominantly clustered in the Mono-like(p<0.05), but TP53^mut samples showed a trend of clustering in the HSPC-like(p<0.1). Adult and pediatric samples with FLT3-ITD were enriched in the mono-like and HSPC-like groups, respectively (p<0.05). Functional enrichment of DEGs between children and adults revealed MHC-I/II related pathways. HLA expression analysis showed significantly higher HLA-I/II expression in pediatric myeloblasts.

Among the >56,000 identified T/NK cells, six subtypes were classified: conventional T, gamma-delta T (gdT), mucosal-associated invariant T (MAIT), proliferating T, double-negative T (dnT), proliferating T and NK. The composition of T/NK differed between pediatric and adult AML. MAIT, gdT, and conventional T cells were higher in children, whereas dnT and NK cells were higher in adults (p<0.05).

The most abundant conventional T cells were sub-classified into 12 CD8+ subtypes and 10 CD4+ subtypes. Different cell compositions were observed among pediatric AML, adult AML, and HD. CD8+ T proportion was found to be comparable between pediatric AML and HD. In CD8+ T, naïve-like T and stemness TCF7+ ZNF683+ tissue-resident memory T cells were significantly enriched in pediatric AML(p<0.01). Conversely, the proportion of stemness LMNA+ TM, and pre-dysfunction GZMK+ CD69hi/CCL3+ T, were higher in adults. Functional enrichment analysis suggested that pediatric CD8+T cells presenting upregulated genes in metabolism and cytotoxic pathways, while adults in apoptosis, autophagy, and TNF signaling.

In CD4+ T cells, significant composition differences were observed between pediatric and adult AML (p<0.05). In detail, CREM+ DUSP4+ dysfunction T and CCR6+RORA+ Th17 were enriched in adults. The scoring analysis revealed a higher stemness phenotype in the pediatric CD4+T cells compared to the adults. Functional enrichment analysis on the DEGs in CD4+T cells demonstrated that upregulated genes in children involved in the T Helper cell differentiation and T cell receptor signaling, while in adults enriched in the autophagy and apoptosis.

NK cells were broadly clustered into circulating NK and immature bone marrow resident NK (CXCR4+). Adult NK composition was significantly different from the pediatric AML and HD (p<0.05), pediatric AML was comparable to HD. Although adult AML showed a higher NK cell proportion (mainly expanded in resident NK cells), the cytotoxic score was found to be lower than pediatric AML(p<0.05). In circulating NK, stemness SELL+ NK and cytotoxic SYNE1+ NK were higher in pediatric AML (p<0.05). Functional enrichment results also showed pediatric AML enriched in cytotoxicity, while adult enriched in autophagy.

Conclusions

Significant differences in the composition of myeloblasts, T, and NK cells between pediatric and adult AML were identified at the single-cell level, suggestive of a more immunoresponsive nature of pediatric AML.