British zyxwvutsrqponml /ournu/ zyxwvutsrqponm o/ Haematology. 1989. zyxwvutsrq 73, 289-295 zyxwvutsrq Detection of monoclonal B lymphocytes in bone marrow and peripheral blood of multiple myeloma patients by immunoglobulin gene rearrangement studies IVAN VAN RIET, CARL0 HEIRMAN, PATRICK LACOR, MARC DE WAELE, KRIS THIELEMANS AND BEN VAN CAMP Department of Haematology and Immunology, Medical School of the Vrije Universiteit Brussel. Laarbeeklaan 101, 1090 Brussels, Belgium Received 31 May 1989; acceptedfor publication 4 July 1989 Summary. To investigate whether B lymphocytes are involved in the malignant cell clone of multiple myeloma (MM), we performed immunoglobulin gene rearrangement analysis of mononuclear cells and separated B lymphocytes, isolated from bone marrow and peripheral blood of MM patients. The B lymphocytes were separated by immunomag- netic beads, coated with an HLA class zyxwvut II specific antibody. Southern blot analysis with a JH probe revealed in the bone marrow of three out of seven patients identical immunoglo- bulin gene rearrangements in the B lymphocytes when compared to the plasma cells. Out of 10 patients, two patients with a high tumour burden were found to have monoclonal B lymphocytes in the peripheral blood. These results suggest that B lymphocytes in the bone marrow are part of the myeloma clone and that they can circulate in the peripheral blood. Although previous studies indicated that the ratio of K to 1 bearing lymphocytes in the peripheral blood can provide evidence for B cell monoclonality. we did not fmd a correla- tion between the results of K/1 analysis and immunoglobulin gene rearrangement. Multiple myeloma (MM) is regarded as a malignant disease originating from plasma cells infiltrating the bone marrow. During the last decade, several studies have been reported suggesting the neoplastic involvement of early B cells, by showing their expression of immunoglobulins (Ig) with the same light chain (Abdou & Abdou, 1975: Sat0 et zyxwvutsr al, 1978: Mellstedt et al, 1976) or the same idiotype (Id) determinants (Kubagawa zyxwvutsrq et al, 1979: Bast et al, 1982) as the monoclonal Ig secreted by the malignant plasma cells. The presence of monoclonal B lymphocytes in the peripheral blood led to the observation that the number of clonotypic B lymphocytes in the blood was associated with the tumour mass (Warner & Krueger. 1978). However, some reports gave a conflicting interpretation to those observations and claimed that the clonotypic reactivity pattern was due to specific binding of serum monoclonal Ig (King & Wells, 1981; Van Camp, 1980). In addition, two studies failed to show a significant number of peripheral blood lymphocytes expressing mono- clonal surface Ig (Pilarski et al. 1984: Gobbi et al. 1984). However, in patients in the plateau phase of their disease, expression of the light-chain isotype concordant with the M component on circulating blood lymphocytes is suppressed Correspondence: Dr I. Van Riet. Department of Haematology and Immunology. Laarbeeklaan 10 1, 1090 Brussels, Belgium. (Van Camp et al. 1981; Joshua et al. 1987). Thus the statement that circulating B lymphocytes are part of the neoplastic clone in MM is still controversial and needs clarification in view of recent attempts to use peripheral stem cells for transplantation in refractory MM patients (Fermand et al, 1988: Laporte et al. 1988). Recently, the analysis of Ig gene rearrangements has allowed the study of monoclonality of B cells at the genetic level, avoiding the potential artefacts of immunophenotyping (Cleary et al, 1984a. b). With this method the plasma cells (Selvanayagam et al, 1988) as well as some of peripheral mononuclear cells (Berenson et al. 198 7) in MM were shown to be monoclonal. In this study, we have examined the Ig gene rearrange- ment of bone marrow and peripheral B lymphocytes, isolated on the basis of HLA class I1 surface expression, in order to examine their oncogenic involvement and their relationship with the myeloma tumour burden. MATERIAL AND METHODS Patient samples. The MM patients who entered this study included newly diagnosed patients and previously treated patients who had clinical signs of progression but who had not undergone any treatment during the preceding weeks. 289