Hepatitis C virus (HCV) seems to be the aetiologic agent of mixed cryoglobulinaemia, and as this 'benign' lymphoproliferative disorder can frequently develop into more aggressive haematological disorders, this study was undertaken to determine the prevalence of HCV infection in non-Hodgkin's lymphomas. 199 unselected subjects treated by three haematological centres in Northeast Italy were investigated for the presence of HCV infection. As controls, the prevalence of HCV infection was determined in a group of patients affected by other haematological malignancies (153 subjects) and in the general population of the same geographical area in the cohort study called the Dyonisos project (6917 subjects). The presence of anti-HCV antibodies was determined by a commercial kit and, in positive cases, by PCR amplification of the 5' untranslated region of the virus. The HCV genotype was also obtained by PCR amplification of the Core region with type-specific primers. The presence of serum cryoglobulins was determined in each case of NHL. HCV infection was significantly (P < 0.00000001) higher in patients with non-Hodgkin's lymphomas (28.0%) when compared with that of the general population (2.9%), and with the group of patients affected by other malignancies (3.1%). The prevalence is particularly high in low-grade (38.4%), as compared with intermediate (11.4%), or high-grade (15.2%) lymphomas. The presence of the virus is significantly (P < 0.000001) associated with the presence of detectable levels of cryoglobulins. On the basis of these findings. HCV seems to play an important role in the development of low-grade non-Hodgkin's lymphomas.

BACKGROUND Several authors have reported on the effectiveness of α‐interferon (IFN‐α) in the treatment of patients with mixed cryoglobulinemia. This prompted the authors to investigate the long term effects of this drug on clinical, hematologic, and virologic parameters in a group of 20 patients (13 women and 7 men) affected by mixed cryoglobulinemia. METHODS In all patients, bone marrow biopsy, phenotyping of marrow cells, and polymerase chain reaction (PCR) immunoglobulin gene rearrangement in peripheral blood lymphocytes were performed before therapy and at the end of the follow‐up. A liver biopsy was obtained in patients with biochemical signs of chronic liver disease. The presence of hepatitis C virus (HCV) RNA in serum was assessed by detection of anti‐HCV antibodies, and by PCR amplification of the 5′ untranslated region of HCV. The HCV genotype was also determined by PCR amplification of the core region of the virus with type‐specific primers. The treatment schedule followed by all patients was 3 million units of recombinant IFN‐α 2b 3 times weekly for 1 year. RESULTS In 6 patients, the marrow histology before therapy showed a massive (more than 50%) monomorphous infiltration by plasmacytoid lymphocytes, indicating the presence of low grade non‐Hodgkin's lymphoma. Anti‐HCV antibodies were present in 19 (95%) subjects, and HCV‐RNA was detectable in all patients. In addition, all patients affected by Type II mixed cryoglobulinemia showed a monoclonal B‐cell expansion in peripheral blood mononuclear cells (PBMC). With therapy, 5 patients (25%) achieved a complete response and 11 patients (55%) a partial response, whereas minor responses were observed in the remaining 4 patients (20%). One of the complete responders and all patients showing partial responses relapsed a few months after therapy withdrawal. At the end of the follow‐up, four patients had obtained a complete remission. Bone marrow examination showed that B‐lymphocytic monoclonal infiltrate disappeared in three patients. Moreover, these three patients had become negative for B‐cell expansion in PBMC. Lack of response, or relapse, was associated with the presence of Type II HCV. CONCLUSIONS HCV may be the cause of mixed cryoglobulinemia. The disease is associated with a high prevalence of bone marrow B‐cell lymphomas. IFN‐α appears to be an effective agent for the treatment of mixed cryoglobulinemia. It also seems able to determine regression of the lymphoproliferative disorder. The HCV genotype appears to be the most important predictive factor for the response to antiviral therapy. Cancer 1996;77:2604‐13.

Clonal expansions of IgM-producing B cells were investigated in 38 patients with a chronic hepatitis C virus infection. Eight patients were affected with type II mixed cryoglobulinaemia (two of whom also had non-Hodgkin's lymphoma and one had Waldenström's disease), one with type III mixed cryoglobulinaemia, one with Waldenström's disease, and 28 with chronic liver disease. To detect the clonal B-cell expansions we used a RT/PCR procedure in which the CDR3/FW4 regions of the IgM heavy chain mRNAs were amplified and resolved in sequencing polyacrylamide gels. Clonal Ig gene rearrangements were detected in all patients with type II mixed cryoglobulinaemia and also at a high frequency (24%) in the HCV-infected patients without cryoglobulinaemia. A polyclonal pattern was present in the patient with type III mixed cryoglobulinaemia and in the 15 normal individuals and 16 age-related patients with HCV-negative alcoholic liver disease which were investigated as controls. No association was found between the presence of a clonal B-cell expansion and age, sex, liver histology, or levels of serum aminotransferase. The serum levels of rheumatoid factor were increased in all patients with a clonal expansion, suggesting that the expanded B-cell clones belong to the rheumatoid factor producing B-cell subset.

Because a close relationship has been established between mixed cryoglobulinemia and hepatitis C virus (HCV) infection, the clinical, histologic, and virologic findings of 31 patients affected by mixed cryoglobulinemia have been determined. HCV infection was investigated by the presence of anti-HCV antibodies and by polymerase chain reaction (PCR) amplification of the 52 untranslated region (52UTR), and the genotype of HCV was also determined according to Okamoto et al (J Gen Virol 73:673, 1992). A bone marrow (BM) biopsy was performed in all patients, and liver and kidney biopsies were performed when indicated. The prevalence of anti-HCV antibodies was high (83.9%); polymerase chain reaction amplification of the 52 untranslated region was positive in 26 subjects (83.9%), and Core region amplification in 26 of 27 subjects (96.2%). A high prevalence of genotype II was found (76.6%). Chronic liver disease was present in 15 (48%) patients. BM biopsy specimens showed the presence of low-grade non-Hodgkin's lymphomas in 12 cases (38.7%), whereas, in 11 patients (35.5%), the BM infiltration was not monoclonal (reactive). Mixed cryoglobulinemia is closely associated with HCV infection. Apparently, only 1 patient was not infected by the virus. Several HCV genotypes are involved in the pathogenesis of mixed cryoglobulinemia. The disease is associated with a high prevalence of low-grade non-Hodgkin's lymphomas.