Type II mixed cryoglobulinaemia as an oligo rather than a mono B-cell disorder: evidence from GeneScan and MALDI-TOF analyses

Objective. To identify and characterize rheumatoid factor (RF)-producing B-cells and cryoprecipitate immunoglobulin (Ig) M in hepatitis C virus (HCV)-positive patients. Methods. We purified and characterized, by peptide mass fingerprinting integrated with an NCBI IgBlast data bank search, the IgM component of cryoprecipitate and analysed the VDJ pattern of bone marrow B-cells by gene scan analysis of 17 HCV-positive patients with type II mixed-cryoglobulinaemia. Results. IgM purified from all of the patients presented an RF specificity. In three of these patients a high and predominant B-cell clone (¸30%) was found in the bone marrow. B-cell-receptor sequences were determined and immunophenotyping of these clones was performed. Peptide masses originating after tryptic digestion of the B-cell-receptor combinatory regions and those originating by tryptic digestion of the cryoprecipitated IgM from the same patient were comparable. In the remaining patients an oligoclonal/polyclonality was found. However, in some of these patients we were able to find peptides that matched with the B-cell-receptor sequences of overexpanded B cells, indicating that, even in the absence of a clear monoclonal expansion, a fraction of total cryoprecipated IgM may derive from overexpanded B-cell clones found in patients’ bone marrow. Conclusions. In the majority of mixed cryoglobulinaemia-HCV-positive patients, both in the serum and in B cells from the bone marrow, an oligoclonal pattern is the main molecular picture. When a monoclonal B-cell clone is found, its B-cell-receptor shows an antigen-binding fragment identical to that of cryoprecipitable RF-IgM. Phenotypically, B cells are CD20-positive but CD5-negative, suggesting that the B-1 B-cell subset is not likely to produce high-affinity IgM-RF molecules. The term cryoglobulinaemia refers to the presence in the serum of one (monoclonal cryoglobulinaemia) or more (mixed cryoglobulinaemia, MC) immunoglobulins (Ig), which precipitate at temperatures below 378C. This is an in vitro phenomenon; the real mechanism(s) of cryoprecipitation remains obscure. Cryoglobulins are classified according to Brouet’s criteria [1]. MC consists of polyclonal Ig G and either polyclonal (type III; MC-III) or monoclonal (type II; MC-II) IgM rheumatoid factors (RFs). MC-II is frequently associated with development of serious vascular, renal and neurological lesions; B-cell clonal expansions are also a frequent laboratory finding. Oligoclonal or monoclonal B-cell expansions are significant features of liver, peripheral blood and bone marrow, and are associated with clinical manifestations and laboratory data [2]. MC-II has been associated with higher risk of non-Hodgkin’s lymphoma (NHL) [3] and, although more controversial, with cirrhosis [4]. Ectopic lymphoid follicles generally express IgM and the limited number of family genes used to construct the antigen-binding fragment (Fab) suggest the possibility that chronic antigenic stimulation contributes to B-cell expansion [5]. Eighty per cent of the monoclonal IgMs found in hepatitis C virus positive patients with a type II mixed cryoglobulinaemia(HCV-MC) patients share a major complementary region named WA. This WA crossing idiotype has been long recognized to be associated with a high degree of RF activity. These antibodies almost invariably express an IgH variable heavy (VH) chain derived from the V1-69 germinal gene and an IgK variable light chain (VL) derived from the V3-20 germinal gene [6]. Approximately 25% of B-NHLs in patients with HCV type II cryoglobulinaemia appeared to arise from WA cross-reactive B cells, and about 70% of light chain of Ig are from V3-20 or V3-15 germline genes [7, 8]. Other VH/VK gene combinations frequently found in MC-associated NHLs were V3-7/V3-15 and V4-59/V3-20 [9]. All this evidence suggests that the malignant cells could derive from the RF-producing B-cells that sustain of the MC syndrome. However, a direct link between clonal B-cell proliferation and production of the IgM component of the cryoprecipitate still remains to be demonstrated. Clinical and epidemiological evidence and laboratory investigations have established the pathogenic role of HCV in MC-II. Furthermore, HCV is the most prevalent hepatotropic virus in