Circulating B Lymphocytes from Patients with Multiple Myeloma Harbour T(4;14) Translocations and Chromosome 13 Deletions.

Abstract

Multiple Myeloma (MM) is clinically characterized by accumulations of plasma cells in the bone marrow (BM). We have identified drug resistant clonotypic B lymphocytes in the peripheral blood of MM patients that have malignant characteristics and the ability to xenograft MM to immunodeficient mice. Using an automated scanning system (Bioview Duet), for blood samples from 72 MM patients, we scanned cytospin slides stained with May-Grunwald Giemsa to identify lymphocytes and determine whether they have the same chromosomal abnormalities that characterize autologous plasma cells. The location on the slide of each morphologically identified cell is recorded during an initial scan, followed by FISH, and examination of the same cells for genetic abnormalities. For this study, we chose probes to detect 1) deletion of chromosome 13 using D13S319 (Vysis), and 2) the t(4;14)(p16;q32) translocation using a dual fusion probe (Vysis). Both abnormalities correlate with adverse prognosis. Lymphocytes from PBMC of healthy donors show 2.8+/−1% of lymphocytes with Ch13 deletion, and less than 1% with t(4;14). An MM PBMC sample was considered to have lymphocyte abnormalities if it scored above a cut off value of 10% for Ch13 deletions and 2% for t(4;14), this is likely to underestimate the extent of chromosomal abnormalities in MM B cells. The number of abnormal lymphocytes in MM PBMC is sufficiently large, and the number of abnormal cells in comparable populations from healthy donors is sufficiently small, that we are readily able to detect significantly increased numbers of abnormal lymphocytes in MM PBMC. We found that peripheral lymphocytes from 19/60 (32%) MM patients have Ch13 deletion (27%) and/or t(4;14) (26%); for those patients with an available BM sample, these same abnormalities were found in their BM plasma cells. Interestingly, intraclonal heterogeneity is apparent in malignant cells from a t(4;14) patient; these plasma cells include a minority population with apparent Ch14 monosomy (23%) and a major population having two copies of Ch14 (77%). This was internally controlled in that for both populations, the plasma cells have two copies of the fused t(4;14) chromosome, and the polymorphonuclear cells on the same slide were normal. One MM patient had Ch13 deletion in 60% of lymphocytes from PBMC, consistent with the 63% of BM plasma cells from this same patient showing Ch13 deletion. For another MM patient known to be t(4;14)+, sorted sIgM+ B cells were analyzed and found to include a small subset with t(4;14) translocations. For 2/6 MM PBMC, abnormalities in Ch19 were detectable in 25–37% of lymphocytes. For mobilized blood autografts, preliminary data indicates the presence of lymphocytic cells with detectable Ch13 deletion. By performing FISH after immunostaining with anti-CD20 of PBMC from MM patients whose bone marrow plasma cells had Ch13 deletion/monosomy, we detected chromosomal abnormalities in CD20+ B cells from MM blood. No abnormalities were detected for CD20+ B cells from healthy donors. In the context of our previous work, this analysis demonstrates that two molecular signatures of MM, clonotypic IgH gene rearrangements and, as reported here, prognostically important chromosomal abnormalities are found among circulating CD20+ B cells from the blood of patients with MM. This work confirms that the MM clone includes circulating B lymphocytes harbouring chromosomal deletions or IgH switch region translocations known to be clinically significant in MM, further implicating them as a source of relapse.