Biologic characteristics of B-lymphoblastic leukemia correspond to immunoglobulin gene diversity [sequencing]

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) 1 and 2 rearrange the immunoglobulin heavy chain (IgH) locus in B lymphoid progenitors, resulting in unique variable (Vh), diversity (D), and joining (Jh) gene rearrangements. To test whether discrete properties of each B-ALL reflect derivation from distinct developmental B cell stages, we assessed the relationship between IgH variable gene (IGHV) characteristics and gene expression. Using targeted, pre-treatment IgH sequencing of 22 patients’ B-ALL, we discovered 2 distinct groups: leukemias with RAG-mediated intra-clonal IGHV sequence diversification (‘diverse’; N=9, 40.9%) and those lacking diversity (‘homogeneous’; N=10, 45.5%). Three patients (13.6%) lacked a dominant IgH sequence. In the ‘diverse’ cohort, we observed extensive subclone evolution arising from Vh gene recombination after neoplastic transformation (median: 482 subclones; range 2-2600). In the ‘homogeneous’ cohort, all IgH clones lacked subclone evolution. The ‘diverse’ cohort demonstrated enriched expression of genes associated with regulation of a hematopoietic stem cell state, while ‘homogeneous’ cases showed enriched expression of genes involving cell fate commitment. Furthermore, distinct IgH clonotypes within a single patient could be individually diverse or homogenous. Single cell analysis indicated that within a single patient, only occasional IgH clonotypes were expressed; IGHV-expressing leukemia cells were characterized by distinctive phenotypes including RAG1 pathway up-expression. In summary, pre-treatment IgH composition reflects the B cell stage at leukemic initiation and provides insights about the biologic mechanisms of genetic diversity in B-ALL. We evaluated pre-treatment bone marrow (BM) and/or peripheral blood (PB) from 22 patients with newly diagnosed B-ALL. Associated data included age, presenting WBC count, immunophenotype, and cytogenetics. We performed targeted IgH NGS on pre-treatment PB for all 22 cases and on BM for the 16/22 for whom BM was available. Fourteen patients with available viably frozen tissue underwent RNA sequencing (RNAseq), and 7 specimens from 5 patients underwent single-cell RNAseq. Chromosome microarray (CMA) analysis was conducted on the 18/22 cases with available B-ALL DNA after IgH NGS.