Genomic landscape of Down syndrome-associated acute lymphoblastic leukemia

Abstract

Trisomy 21, the genetic cause of Down syndrome (DS), is the most common congenital chromosomal anomaly. It is associated with a 20-fold increased risk of acute lymphoblastic leukemia (ALL) during childhood and results in distinctive leukemia biology. To comprehensively define the genomic landscape of DS-ALL, we performed whole-genome sequencing and whole-transcriptome sequencing (RNA-Seq) on 295 cases. Our integrated genomic analyses identified 15 molecular subtypes of DS-ALL, with marked enrichment of CRLF2-r, IGH::IGF2BP1, and C/EBP altered (C/EBPalt) subtypes compared with 2257 non-DS-ALL cases. We observed abnormal activation of the CEBPD, CEBPA, and CEBPE genes in 10.5% of DS-ALL cases via a variety of genomic mechanisms, including chromosomal rearrangements and noncoding mutations leading to enhancer hijacking. A total of 42.3% of C/EBP-activated DS-ALL also have concomitant FLT3 point mutations or insertions/deletions, compared with 4.1% in other subtypes. CEBPD overexpression enhanced the differentiation of mouse hematopoietic progenitor cells into pro-B cells in vitro, particularly in a DS genetic background. Notably, recombination-activating gene-mediated somatic genomic abnormalities were common in DS-ALL, accounting for a median of 27.5% of structural alterations, compared with 7.7% in non-DS-ALL. Unsupervised hierarchical clustering analyses of CRLF2-rearranged DS-ALL identified substantial heterogeneity within this group, with the BCR::ABL1-like subset linked to an inferior event-free survival, even after adjusting for known clinical risk factors. These results provide important insights into the biology of DS-ALL and point to opportunities for targeted therapy and treatment individualization.

Graphical abstract

Figure 1.

Transcriptomic and genomic landscape of DS-ALL. (A) Subtype classification of 295 DS-ALL cases with available RNA-Seq or paired tumor-germline WGS data. Each column represents a patient, and each row represents a feature, including sex, age, availability of data, rearrangements, and expression of CRLF2, CEBPD, and IGF2BP1. The patients are ordered according to subtypes. In 2 cases with CEBPD overexpression, chromosomal rearrangements of CEBPD locus to intergenic regions of 9p13.2 (between PAX5 and ZCCHC7) and 6q25.3 (∼380 kb upstream ARID1B) were identified. As no fusion transcript was identified by RNA-Seq, these rearrangements are denoted as 9p13.2::CEBPD and 6q25.3::CEBPD. (B) Gene expression of 249 DS-ALL cases visualized by UMAP. Top 100 genes with the highest median absolute deviation were used to generate this 2-dimensional UMAP. Subtypes are shown in different colors. (C) Genomic alterations in 244 DS-ALL cases. The cases are ordered by subtypes, as indicated above the panel. Only genes significantly altered in DS-ALL are included.

Figure 2.

Genomics of IGH::IGF2BP1 and C/EBPalt DS-ALL. Positions and rearrangement partners of (A) IGF2BP1 and (B) CEBPD rearrangements, as identified by paired WGS data. For IGH::IGF2BP1 and IGH::CEBPD rearrangements, the specific IGH gene segments that CEBPD rearranged to are shown, all of which are IGHJ gene segments. (C) Hierarchical clustering identified 8 cases with similar gene expression signature to CEBPD-r DS-ALL, indicated as C/EBPalt (other) with purple color in the top bar denoting sample subtypes. (D) Mapping of CEBPA and CEBPE alterations in 5 C/EBPalt cases. (E) Comparison of expression of CEBPA, CEBPE, and CEBPD genes among DS-ALL subtypes. The 5 cases with CEBPA- or CEBPE-related genomic alterations in panel D are marked by numbers. (F) Lollipop plot showing the location of FLT3 alterations in C/EBPalt DS-ALL. Most of the FLT3 alterations are in the juxtamembrane domain or the second tyrosine kinase domain. (G) FLT3 is highly expressed in the C/EBPalt subtype.

Figure 3.

CEBPD overexpression and a DS genetic background contribute to differentiation to the pro-B stage demonstrated using scRNA-Seq of CEBPD and control transduced WT and Dp16 murine cells. (A) UMAP projection and clustering of cells from all 4 conditions. (B) Expression of hematopoietic differentiation marker genes assigned the 13 clusters to 4 cell types. (C) UMAP projection and cell differentiation stage of the 4 conditions. (D) The frequencies of each cell type in the clusters across the 4 conditions. (E) Cell cycle analysis of the cells and the percentages of pro-B cells in each cell cycle phase across the 4 experimental conditions.

Figure 4.

Heterogeneity of CRLF2-r DS-ALL. (A) Unsupervised hierarchical clustering of 130 CRLF2-r DS-ALL samples with RNA-Seq data identified a unique group of cases with BCR::ABL1-like gene expression signatures. BCR::ABL1-like classification by PAM based on non–DS-ALL data is indicated by red bars above the heatmap. (B) Projecting the BCR::ABL1-like and non–BCR::ABL1-like CRLF2-r subtypes to UMAP shows that BCR::ABL1-like CRLF2-r cases cocluster with BCR::ABL1 and non-CRLF2-r BCR::ABL1-like, and are distinct from non–BCR::ABL1-like CRLF2-r. (C) Genomic alterations in BCR::ABL1-like and non–BCR::ABL1-like CRLF2-r subgroups. Genes altered in >6% of CRLF2-r DS-ALL are shown.

Figure 5.

Comparisons between DS-ALL and non–DS-ALL. (A) Frequencies of genetic subtypes of DS-ALL and non–DS-ALL. (B-C) Comparison of the frequencies of gene alterations in DS-ALL and non–DS-ALL in (B) all the subtypes and (C) CRLF2-r only. A total of 51 genes altered in more than 3% in either DS-ALL or non–DS-ALL data are compared and only genes with nominal P < .05 are shown. Comparisons were also performed within ETV6::RUNX1 and high hyperdiploid subtypes and no gene with Bonferroni corrected P < .05 was identified.

Figure 6.

Mutation signature of DS-ALL. (A) SNV mutation signature of DS-ALL subtypes. (B) Number of RAG-mediated structural alterations identified in DS-ALL subtypes.

Figure 7.

Kaplan-Meier curves. Kaplan-Meier estimates of the (A) EFS and (B) OS according to major subtypes in DS-ALL. Subtypes with <5 cases are included in “Others.” Kaplan-Meier estimates of the (C) EFS and (D) OS of BCR::ABL1-like and non–BCR::ABL1-like subtypes in CRLF2-r DS-ALL.