Children with Acute Lymphoblastic Leukemia and Early Thrombosis Have Dysregulated Coagulation-Related Genes and Pathways

Introduction Thrombo-embolic events (TE) are a frequent complication of childhood acute lymphoblastic leukemia (ALL). Disease-specific mechanistic insights governing the development of TE in this disease remain unclear. Overexpression of coagulation-related genes (coagulome) and pathways have been identified in cancer-induced TE. We investigate whether coagulome dysregulation in newly-diagnosed children with ALL is associated with TE development.

Methods We included patients aged 1-21 years old with newly-diagnosed ALL from September 3, 2015, to June 30, 2021, with available RNA-sequencing (RNAseq) data at diagnosis, either enrolled on the Dana-Farber Cancer Institute (DFCI) ALL Consortium Protocol 16-001 or treated following the DFCI 11-001 protocol. RNAseq was performed through an optional study performed as part of the DFCI 16-001 clinical trial for patients who consented to it, or through the research oncogenomic program of the province of Quebec. The primary outcome was the occurrence of TE during ALL therapy requiring medical intervention (grade ≥2 as per the CTCAE version 5). TE were classified as early thrombosis (ET) if they occurred within 6 weeks from treatment start. Patients' characteristics were compared using Chi-Square or Fisher's exact test. Differential gene expression (DE) was performed to compare children with and without TE, adjusted for ALL phenotype (B- or T-cell lineage). The dysregulated genes were compared to a customized panel of 174 genes known to be implicated in cancer-related TE (coagulome and podoplanin) and in coagulation pathways. A secondary analysis, determined a priori, stratified children between ET and no TE, based on the hypothesis that the leukemia environment might predominantly affect early complications. Gene set enrichment analyses (GSEA) were performed on KEGG and gene ontology (GO) databases from the molecular signatures database (MSigDB). DE was considered for absolute fold change (FC) ≥2, and benjamini-Hochberg adjusted p-values (adj p) <0.05.

Results We included 274 ALL patients (median age: 6.7 years [interquartile range, IQR: 3.7-12.0 years], 54% male, 81% B-ALL) of whom 49 (17.9%) experienced a TE (including 21 ET) at a median of 76 days (IQR: 27-155 days) following ALL diagnosis. Patients with TE were more likely to be ≥10 years old (57.1% vs. 31.6%, p<0.001) compared to those without TE. Sex, immunophenotype, mediastinal mass, central nervous system disease (CNS) involvement at diagnosis and risk status were otherwise not significantly different among the two groups.

Three coagulation pathway-related genes were dysregulated in children with TE compared to those without TE. Glycoprotein 9 (GP9) and platelet factor 4 (PF4), implicated in von Willebrand-dependent platelet adhesion and platelet aggregation, were upregulated (log2FC = 2.1 and 1.5, respectively). Metalloproteinase-2 (MMP2), involved in collagenolysis and thrombus degradation, was downregulated (log2FC = -1.9). The KEGG olfactory transduction pathway was downregulated in the group with TE (normalized enrichment score (NES) = 1.55, adj p<0.001).

In the secondary analysis stratified by timing of TE, patients with early TE were more likely to be ≥10 years (80.9% vs. 31.6%, p<0.001), have T-ALL (38.1% vs. 16.9%, p=0.002) and be CNS-positive at diagnosis (52.4% vs. 22.7%, p=0.004). Patients with ET showed overexpression of the GP9, PF4, integrin subunit beta 3 (ITGB3) and clusterin (CLU) genes involved in platelet aggregation and adhesion (log2FC = 3.3, 3.7, 2.7 and 1.6, respectively). The following GO pathways were significantly upregulated: blood coagulation (NES=1.73, adj p<0.001), platelet activation (NES=2.21, adj p<0.01), and platelet alpha granules (NES=2.48, adj p<0.001) while the KEGG olfactory transduction pathway was downregulated (NES=-1.70, adj p<0.001). Dysregulation of the olfactory transduction pathway has previously been described in cancer-associated TE in adults.

Conclusion Our study identified coagulation-related genes and pathways that might be drivers of leukemia-induced thrombosis. Early TEs in childhood ALL were associated with significant upregulation of coagulation and platelet activation pathways, suggesting a role of the leukemia microenvironment in TE development. Additional studies are required to validate these observations that could be incorporated in future algorithms to predict TE risk in children with ALL.

Silverman:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees. Tran:Jazz: Consultancy, Research Funding; Servier: Consultancy, Research Funding.