Bcl-2 rearrangement in patients with chronic hepatitis C associated with essential mixed cryoglobulinemia type II

Hepatitis C virus (HCV) infection is found in 80% to 90% of patients with essential mixed cryoglobulinemia (EMC) type II.1,2 EMC type II is characterized by polyclonal IgG and monoclonal IgMk.1,3 Monoclonal IgMk is secreted by clonal B lymphocytes, and this may suggest that low-grade non-Hodgkin lymphoma (NHL) is the underlying disorder of EMC.4-6 

The association between EMC, HCV, and NHL raises the possibility that HCV may be involved in the pathogenesis of lymphoma. Several epidemiological studies conducted in Italy, Japan, and North America showed that the prevalence of HCV seropositivity or viremia (RNA) is significantly increased in patients with B-NHL (but not in patients with Hodgkin disease or T-cell lymphoma) relative to the general population.7-9 

Rearrangement of bcl-2 is found classically in follicular, low-grade NHL that has a prolonged, slowly progressive clinical course, often terminating in transformation to high-grade lymphoma.10Recently, we reported a patient with EMC and monoclonal B-cell proliferation who carried the translocation 14:18 (q32:q21), involving IgH/bcl-2, during the “benign” phase of her disease. Clinical progression was associated with a second genetic aberration involving the c-myc oncogene.11 Later, it was also shown that strong bcl-2 expression can be found in B lymphocytes obtained by liver and bone marrow biopsies of patients with EMC.6 

In the current study we extended our efforts to study the JH/bcl-2 translocation in patients with HCV. We used fluorescence in situ hybridization (FISH) to analyze the occurrence of bcl-2 rearrangement (JH/bcl-2 translocation) in leukocytes of patients with chronic liver disease (CLD) caused by HCV infection with or without EMC and compared it with the occurrence in patients with non-HCV CLD and in healthy subjects.

Patients were randomly recruited from liver clinics in 4 university-affiliated hospitals in Israel (Sapir Medical Center, Rabin Medical Center, Soroka Hospital, and Hadassah University Hospital). They were divided into 3 study groups: patients with HCV-related CLD and EMC (HCV+EMC+); patients with HCV-related CLD without EMC (HCV+EMC−); and a randomly selected group of patients with CLD without HCV or EMC (HCV−EMC−). In addition we studied 10 healthy persons as controls.

All patients were studied through the FISH method (detailed in12). A bcl-2 probe, a 4.2-kb HindIII genomic fragment, and a heavy-chain JH probe containing a 3.5-kbHindIII–EcoRI fragment (provided by Y. Tsujimoto) were applied.

Microscopy was carried out using an Olympus BHS (Olympus Optical, Tokyo, Japan), with fluorescence equipment and filter for simultaneous viewing of DAPI, FITS, and Texas red (Omega). We attempted to analyze 100 cells from each slide. FISH tests were performed blindly, and the laboratory team was unaware of the clinical status.

The cutoff rate for the diagnosis of JH/bcl-2 translocation was determined as the average number of control cells with the juxtaposition of JH and bcl-2 (6.1%) plus 3 SD. Thus, a patient was considered to have a translocation when 13.5% or more cells had juxtapositions of JH and bcl-2.

Five patients randomly selected from among those with JH/bcl2 translocation by FISH were studied by the classic cytogenetic methods, and 3 of them were studied by FISH for metaphases with painting probes. For this latter method, the following VYSIS probes were used: WCP 14 spectrum orange (no. 33120014) and WCP 18 spectrum green (no. 33122018).

HCV RNA detection was determined by RT-PCR assay, performed using the Amplicor kit (Roche, Diagnostic System, Branchburg, NJ).

Analysis and characterization of cryoglobulins were performed according to the method of Polzien et al.13 

The 2-sample t test and the nonparametric (Wilcoxon) test were applied for testing differences between the study groups for quantitative parameters. The multiple comparisons (Duncan method) tests were applied for testing quantitative parameters between the study groups. All tests were 2-tailed, and P < .05 or less was considered statistically significant. Data were analyzed using the SAS software (SAS Institute, Cary, NC).

The current study verifies our original hypothesis of bcl-2 rearrangement in patients with EMC associated with HCV, as shown in Table 1. Thirteen of 15 (86%) patients with HCV+EMC+ CLD (group A) had evidence of JH/bcl-2 translocation in 18% to 79% of their peripheral blood leukocytes. This was significantly higher than the rate in patients with HCV+EMC− CLD (group B), of whom 2 of 12 (16%) carried the JH/bcl-2 translocation in their leukocytes (P < .001). This latter rate (16%) in HCV+EMC− patients is significantly higher than the rate in patients with CLD without HCV and in healthy controls (P < .001 and P < .001, respectively). In group C (CLD without HCV infection), no patient had translocations above the cutoff level. Most HCV+EMC+ patients had a high fraction of leukocytes carrying the translocation (mean, 40%; Table 1).

Five patients randomly selected from group A (HCV+EMC+) who had the JH/bcl-2 translocation were also studied by cytogenetic methods, and 3 of them were analyzed by FISH for metaphases with painting probes. The t(14:18) was detected in 1 patient by each method (patients A4 and A1, respectively).

In this study we found that HCV-related EMC is associated with t(14:18). Two additional groups are studying the prevalence of t(14:18) in HCV-related lymphoproliferation using the polymerase chain reaction (PCR). Zignego et al14 found t(14:18) in 71% of their EMC+ HCV+ patients and in 26% of their HCV+ EMC− patients. Only 4% of patients with HCV− CLD were found to carry the translocation. These results are similar to our findings and lend further support to our observation by a different molecular technique. Zuckerman et al15 also used the PCR method but found bcl-2 rearrangement in only 19% of their EMC+ HCV+ patients and in 14% of their HCV+ EMC− patients. The causes of these variances are uncertain and may be related to technical or demographic differences. Nevertheless, all 3 studies clearly suggest that the bcl-2 rearrangement may have an important role in the pathogenesis of lymphoproliferation related to HCV.

Bcl-2 rearrangement is probably associated with the extended survival of lymphocytes in the benign phase of EMC. During their prolonged survival, these cells may undergo additional genetic changes (second hit) that may induce a transformation to overt NHL. Additional follow-up is needed to determine whether NHL will develop in these patients and to evaluate the effect of antiviral treatment on bcl-2 rearrangement.

The mechanism of HCV-induced lymphoproliferation is still debated. HCV has been reported to induce clonal B-cell expansions in the peripheral blood of infected patients.16 HCV sequences were also detected in pathologic lymph node biopsies in 13 of 34 patients with NHL.17 Furthermore, Sansonno et al18 recently demonstrated the presence of HCV-associated proteins within lymphoma cells. However, HCV RNA sequences cannot be integrated into the host genome, and the mechanism of inducing clonality remains unresolved. A different mechanism may involve chronic antigenic stimulation induced by HCV infection leading to B-cell proliferation and later to the emergence of a clone that has a proliferative advantage and, after additional genetic hits, transforms to NHL.19 Thus it remains to be elucidated whether HCV activates its oncogenic potential by indirect mechanisms or uses antiapoptotic pathways directly.