ROR1 Is Expressed on the Surface of Non-Neoplastic Human B-Lymphocyte Precursors (Hematogones) and on a Subset of B Acute Lymphoblastic Leukemia

Abstract 2735

ROR1 is a putative receptor tyrosine kinase that is expressed during embryogenesis, on chronic lymphocytic leukemia (CLL) cells, but not on normal mature lymphocytes, including normal CD5+ B cells (Fukuda et al PNAS 2008; 105:3047). ROR1 binds Wnt5a, and this ligand enhances the survival of CLL cells in vitro, suggesting that ROR1 functionally contributes to leukemogenesis. Additional evidence for the importance of ROR1 function in leukemia is the noted decrease in survival of B acute lymphoblastic leukemia (B-ALL) cells in vitro after RNAi targeting of ROR1 (Tyner et al. PNAS 2009; 106:8695). To further explore the significance of ROR1 in B-ALL, we analyzed 30 cases of B-ALL for surface expression of ROR1 via multiparameter flow cytometry using a fluorochrome-conjugated mAb specific for ROR1. Only two of 30 cases of B-ALL showed uniform surface ROR1 staining with a mean fluorescence intensity (MFI) that was 6.2 or 3.6 times greater than that of B-ALL cells stained with a control mAb. The other 28 B-ALL cases showed a mean MFI ratio of 1.4 (S.D. = 0.4). The two ROR1+ cases shared distinctive immunophenotypic and cytogenetic changes consistent with a more mature B-ALL subtype. We also evaluated marrow from patients recovering from chemotherapy or hematopoietic stem-cell transplantation and found that non-neoplastic B-lymphocyte precursors (BLP, hematogones) also expressed surface ROR1. These BLP were positive for CD10, CD19, CD38, and dim CD45 with variable expression of CD20, CD34, TdT, and cytoplasmic immunoglobulin. While the surface antigen phenotype of B-ALL is similar to BLP, flow cytometry can distinguish B-ALL from BLP because B-ALL shows more uniform antigen expression, lacking the continuum of maturation stages present in BLP. Also, B-ALL usually has aberrant antigen-expression intensity or aberrant antigen co-expression such as terminal deoxynucleotidyl transferase (TdT) plus CD20 on the same cell. Of 22 marrow samples tested with 1–20% hematogones, all had CD10+, CD19+, dim CD45+ cells that expressed ROR1 with a mean MFI ratio of 4.9 (S.D. 2.0). Further study found ROR1 was restricted to a subset of CD10, CD19, and low-level CD45 cells that lack expression of CD34 or TdT. This subset is consistent with an intermediate stage of B-cell maturation, possibly associated with B-lymphoid positive and negative selection. In contrast, CD10+, CD19+ germinal center B cells from reactive lymph nodes were negative for surface ROR1 (N=4). This pattern of ROR1 expression on BLP and a subset of distinctive B-ALL suggests that surface expression of ROR1 occurs during a discrete stage of B-cell maturation in the marrow that may be expanded during recovery from myeloablative therapy and/or hematopoietic stem cell transplantation.