Leukemia (1998) 12, 121–127 1998 Stockton Press All rights reserved 0887-6924/98 $12.00 REVIEW Multiple myeloma: the cells of origin – A two-way street JR Berenson, RA Vescio and J Said West Los Angeles VA Medical Center, UCLA School of Medicine, Los Angeles, CA, USA Multiple myeloma results from an interplay between the mono- earlier precursor cells may be responsible for the proliferation clonal malignant plasma cells and supporting nonmalignant of the malignant population. The presence of a circulating cells in the bone marrow. Recent studies suggest that the final transforming event in this B cell disorder occurs at a late stage tumor component without obvious plasma cell morphology of B cell differentiation based on the characteristics of the also suggests that less mature lymphocytes may be part of the immunoglobulin genes expressed by the malignant clone as clone as well, which could explain the dissemination of the well as surface markers present on the tumor cells. Recently, disease throughout the bone marrow.4 In addition, evidence an increasing pathogenic role in this malignancy by the nonma- for tumor cells at even earlier stages of hematopoietic differen- lignant cells in the bone marrow has been suggested by several tiation came from studies showing the high rate of acute non- studies. Specific infection of these supporting cells by the lymphoblastic leukemia in these patients and the presence of recently identified Kaposi’s sarcoma-associated herpes virus 5,6 (KSHV) suggests a novel mechanism by which this nonmalig- non-lymphoid surface markers on malignant plasma cells. A nant population may lead to the development of this B cell variety of molecular biological techniques have subsequently malignancy and support its growth. been used to characterize further the cell types which are part Keywords: multiple myeloma; immunoglobulin genes; Kaposi’s of the malignant clone in this B cell malignancy. sarcoma-associated herpes virus Immunogenotypes Introduction Because of the monoclonal nature of the Ig synthesized by the malignant cells, the genes, which lead to the production of Multiple myeloma is characterized by the accumulation of this protein, can be used as molecular markers capable of malignant plasma cells in the bone marrow compartment.1 identifying surface markers on the malignant cells. In addition, These terminally differentiated B-lymphocytes all produce an specific characteristic changes in these genes occur at differ- identical immunoglobulin (Ig) known as a monoclonal pro- ent stages of B cell differentiation; and thus allow determi- tein, the laboratory hallmark of this malignancy. Although the nation of which cell types in this lineage are part of the predominant cell type in the bone marrow of these patients tumor clone. has the characteristics of a plasma cell, the low proliferative Each antibody-producing B cell produces a single type of activity of these cells has raised the possibility that less mature antibody. Early in normal B cell development, rearrangement B-lymphocytes represent the proliferating population, which of four separate gene segments leads to the development of give rise to the overabundant malignant plasma cell. Recent the heavy chain portion of a unique functional antibody.7 advances in molecular biological techniques have led to the These gene segments encode the constant region which deter- determination of the stage of B cell differentiation at which mines the class of antibody (eg C␮ for IgM, C␥ for IgG, C␣ this disease begins. for IgA), and three other gene segments which encode the The tumor cells which reside within the bone marrow are variable region of the heavy chain; variable (VH), diversity (D), 7–10 supported by a nonmalignant population of stromal cells and joining (JH). Since these latter three joined segments which produce cytokines that enhance myeloma cell growth encode for the specific antigen recognition site of the final and prevent apoptosis. Moreover, the tumor cells themselves antibody, the recombination of genes which comprise this orchestrate the production of cytokines by these nonmalignant region must be unique to each antibody-producing cell. Thus, bone marrow cells which can lead to a further increase in specific changes in these genes occur during the maturation tumor cell burden. Recent demonstration of the Kaposi’s sar- of the antibody-producing B cell. Most of a single functional coma-associated herpesvirus in some of these nonmalignant heavy chain variable region is encoded by one of ෂ50 func- cells provides an additional novel mechanism by which the tional VH genes whereas shorter stretches of amino acids are bone marrow microenvironment may support this B cell derived from one or more D and one of six functional JH malignancy. genes.7–10 In addition, increased antibody specificity is accomplished by the addition of non-germline nucleotides (N segments).11 Once a functional heavy chain rearrangement The malignant cell of origin has occurred, the ␬ (and, if unsuccessful, ␭) light chain 12 undergoes a similar rearrangement of VL,JL, and C genes. The low proliferative rate of the phenotypically identified Additional specificity of the antibody is rendered by the malignant cell2 and inability of these cells to sustain tumor mutation of nucleotides within the specific areas of the vari- growth in vivo as demonstrated by kinetic studies3 imply that able region which bind antigen directly known as comp- lementarity determining regions (CDRs) leading to enhanced avidity of the antibody for antigen.13 This process of somatic Correspondence: JR Berenson, West Los Angeles VA Medical Center, UCLA School of Medicine, 11301 Wilshire Blvd (111H), Los Angeles, mutation occurs late in B cell development in germinal cen- CA 90073, USA; Fax: 310 268 4908 ters. Following the secretion of a functional active antibody, Received 20 November 1997; accepted 21 November 1997 this process ceases within this terminally differentiated B cell. Review JR Berenson et al 122 In multiple myeloma, the properties of the Ig genes allow of normal plasma cells have not been completed, it is precise determination of the stage of B cell development dur- unknown whether this increased frequency of D-D fusion and ing which malignant transformation occurred. Specifically, lack of N-region addition is specific to myeloma. Use of spe- comparison of the myeloma VH gene sequence to the most cific JH and D genes is quite comparable to usage in mature homologous germline gene has shown marked somatic B-lymphocytes further supporting the late B cell origin of mutation (median 8%) of this gene segment.14 This frequency myeloma. of somatic mutation is only found in the most differentiated B cells after antigenic stimulation has occurred. In further sup- port of an antigenically driven process leading to these malig- Immunophenotype nant clones, analysis of regions which bind antigen within these genes, the CDRs, demonstrates a marked predilection Early studies using aneuploidy as a basis of malignant plasma for somatic mutations compared to the other parts of the gene cell determination suggested the existence of myeloid, mega- which are primarily responsible for maintaining the structural karyocytic, and T cell markers on the surface of the malignant integrity of the molecule, the so-called framework regions clone in myeloma.4 However, the use of these so-called ‘lin- (FRs).15 Moreover, in antigenically driven cells, mutations eage-specific markers’ was problematic since it was learned occurring within the CDRs should be more often associated that normal plasma cells could express these same antigens.32 with a change in the encoded amino acid compared to those The presence of a unique molecular marker, the Ig gene changes occurring in the FRs. Indeed, the ratio of replacement expressed by the malignant clone, has allowed a more precise to silent mutations is double in the CDRs compared to the FRs determination of surface markers present on the malignant in multiple myeloma.14 clone. Importantly, analysis of multiple clones shows no clonal diversity in VH genes from myeloma patients, and determi- nation of these sequences during the course of their disease CD34 shows no clonal evolution.14,16,17 These results differ from other B cell tumors originating in germinal centers at earlier CD34 is expressed on early hematopoietic precursors includ- stages of B cell differentiation.18 Analysis of CDR3 genes com- ing the pluripotent stem cell.33 However, some B cells also 34 prised of the D, JH and N segments further supports the high express this surface marker. Reinfusion of hematopoietic rate of somatic mutation with a high replacement to silent cells selected for CD34 can lead to rapid and sustained ratio.19 These segments also show a lack of clonal diversity.19 engraftment following myeloablative chemotherapy.35 Since Thus, these results suggest that the final oncogenic event in we and others had previously demonstrated the presence of myeloma occurs very late in B cell differentiation. Although tumor cells in not only the bone marrow but also the blood some studies have suggested the existence of a pre-class of myeloma patients, it became of both biological and clinical switched (C␮-containing) monoclonal cell in this disease,20–22 importance to determine whether any malignant cells the frequency of these cells and their contribution to the expressed this stem cell marker in myeloma patients. Initially, malignant process has not been established. Using colony CD34+ cells were purified from myeloma patients’ bone mar- hybridization techniques, our laboratory has not been able to row using a combination of an immunoadsorption column demonstrate C␮-containing clonal cells in myeloma.23 and flow sorting. Using a sensitive PCR-based assay with pri- mers derived from the unique CDRs expressed by the patient’s malignant clone, no tumor cells were detected in the CD34- 36 Use of specific VH, D, and JH genes expressing population. Several other groups have confirmed this initial report37,38 although two reports have suggested the + 39,40 The approximately 50 functional VH genes have been divided existence of a CD34 tumor cell in myeloma.