Biologic Relevance of Variable Immunoglobulin Gene Diversity in B-Lymphoblastic Leukemia

Abstract

Background: It remains unclear whether the variable biologic features of B-lymphoblastic leukemia (B-ALL) reflect distinct cells of origin in the hierarchy of early B cell development. Recombination activating genes (RAG) rearrange the immunoglobulin heavy chain (IgH) locus in B lymphoid progenitors, resulting in unique variable (Vh), diversity (D), and joining (Jh) gene rearrangements. In B-ALL, dominant IgH sequences are used for clone detection and surveillance. Further, subclone diversity resulting from RAG-mediated Vh gene switching is a prevalent feature of many B-ALLs, but its biologic basis has not been determined. To test whether discrete properties of each B-ALL reflect derivation from distinct B cell stages, we assessed the relationship between IgH variable gene (IGHV) characteristics and gene expression. Methods: We used targeted IgH sequencing (Illumina MiSeq) to identify dominant IgH clones and to assess the extent of IgH subclone diversification in pre-treatment specimens from 22 patients with B-ALL. We compared the gene expression profile (GEP) of cases with Vh-switch-mediated subclone diversity to those without. We then used single-cell RNA sequencing (scRNAseq) of 5 patients' B-ALL to test whether the capacity to undergo IgH diversification is patient-specific or clone-intrinsic, and we characterized variation in GEP between intrapatient IgH clones. Results: We discovered that each B-ALL with at least 1 dominant IgH clone demonstrates either minimal or extensive RAG-mediated subclone evolution. Gene set enrichment analysis (GSEA) revealed that genes associated with distinct B cell developmental stages distinguish these 2 groups. We detected dominant IgH clones in 19 of 22 patients (86.4%). In patients with 'diverse' B-ALL characterized by RAG-mediated intra-clonal diversification (N=9; 40.9%), we observed extensive subclone evolution arising from Vh recombination (median: 482 subclones; range: 2-2600) and enriched expression of genes associated with regulation of a hematopoietic stem cell state. In those without diversity (N=10; 45.5%) - the 'homogeneous' cohort - leukemia cells showed enriched expression of genes involving cell fate commitment. GSEA further revealed biologically distinct phenotypes between groups, as reflected in enrichment in the diverse cohort of Hallmark gene sets involving key cellular metabolic pathways including oxidative phosphorylation and glycolysis. Furthermore, distinct dominant IgH clones from a single patient could be individually diverse or homogenous with respect to Vh-switch-mediated subclone evolution. scRNAseq revealed that dominant IgH clones inconsistently express IGHV mRNA and differ from one another in their gene expression. In 4 of 5 patients, we detected IgH complementarity-determining region 3 (CDR3) mRNA expression from at least 1 clone. The GEP of IGHV-expressing leukemia cells was clearly resolved from other B-ALL cells in 3 of these 4 cases, with distinctive gene expression characterized by RAG1 pathway up-expression. Conclusions: Tumor evolution requires genetic diversity and is a fundamental property of cancer. Using primary B-ALL specimens, we assessed RAG-mediated intraclonal IgH variation to characterize the extent of diversity in B-ALL and to discover how it relates to fundamental biologic features of this common pediatric malignancy. We found that B-ALL may be divided into 2 groups: leukemias with RAG-mediated intra-clonal IGHV sequence diversification ('diverse') and those lacking diversity ('homogeneous'). GEP suggests that the difference in diversity of the IGHV repertoire reflects the B cell stage at leukemic initiation: B-ALL specimens with subclone diversity had a GEP suggestive of an earlier differentiation state compared to specimens with a homogenous IGHV repertoire. Furthermore, we discovered that a single leukemia can be simultaneously composed of both genetically stable and genetically diverse clonal populations. Our single-cell GEP data reveal that: 1) even within a single patient's leukemia, genetically-distinct clonal populations demonstrate a distinct profile of mRNA expression, and 2) B-ALL cells which express IGHV mRNA are characterized by greater expression of RAG pathway-related genes. Our findings suggest that variability in IGHV diversification in B-ALL is a useful marker of biological properties related to the B cell stage of origin.