Patrizio Caini

Prevalence of bcl-2 Rearrangement in Patients with Hepatitis C Virus–Related Mixed Cryoglobulinemia with or without B-Cell Lymphomas

Context Rearrangement of bcl-2 has an antiapoptotic effect and has been implicated as a potential cause of benign lymphoproliferation (causing mixed cryoglobulinemia) and B-cell lymphoma. Mixed cryoglobulinemia is strongly associated with hepatitis C virus (HCV) infection. Contribution In patients with HCV-associated chronic liver disease, bcl-2 rearrangement occurred significantly more often in patients with chronic HCV infection and mixed cryoglobulinemia than in HCV-infected patients without mixed cryoglobulinemia; it also occurred in three of four patients with B-cell lymphoma. Transient suppression of HCV in two patients was associated with remission of clinical manifestations of mixed cryoglobulinemia. Implications Viral induction of gene sequence translocations may help explain some benign and malignant lymphoproliferative disorders. The Editors Mixed cryoglobulinemia is a distinct syndrome clinically characterized by purpura; weakness; arthralgia; and such conditions as membranoproliferative glomerulonephritis, peripheral neuropathy, skin ulcers, and diffuse vasculitis (1, 2). Cryoprecipitable immune complexes, specifically mixed (IgG-IgM) cryoglobulins, are the serologic hallmark of the disease. Immunoglobulin Gs are the autoantigens, and IgMs with rheumatoid factor activity are the autoantibodies. Mixed cryoglobulinemia is classified as type II or type III according to the presence of polyclonal or monoclonal IgMs (3, 4). Because expansion of rheumatoid factorproducing B cells is the underlying disorder of mixed cryoglobulinemia, this condition is considered a benign B-cell lymphoproliferative disease. Type II and III mixed cryoglobulinemia are similar in terms of organ involvement and clinical course, except that type II disease may evolve into cancer. Type II mixed cryoglobulinemia is often observed in conjunction with bone marrow findings consistent with indolent B-cell lymphoma (5-9) and evolves to frank B-cell malignancy in about 10% of cases (10). A strong association between mixed cryoglobulinemia and infection with hepatitis C virus (HCV), a hepatotropic and lymphotropic virus (10, 11), has been shown. A pathogenetic role of chronic infection with HCV in mixed cryoglobulinemia has been suggested. The mechanisms involved in benign lymphoproliferation of mixed cryoglobulinemia and its evolution to lymphoma remain unknown. However, rearrangement of the antiapoptotic B-cell lymphoma/leukemia 2 (bcl-2) genethe t(14;18) translocationis suggested to play a role in the pathogenesis of HCV-associated mixed cryoglobulinemia (12, 13). The t(14;18) translocation, the most frequent genetic aberration in human lymphoma (14, 15), may be favored by sustained, strong antigenic stimulation (16-18). As a result of bcl-2 rearrangement, the bcl-2 gene on chromosome 18q21 is coupled with the immunoglobulin heavy chain gene (IgH) on chromosome 14q32 by a process frequently involving IgH joining segments (JH) (Figure 1, top). At the junction of the two genes, insertions of variable lengths (N segments) due to random addition of nongermline nucleotides result in a DNA pattern that is clone specific (19, 20). As a consequence of this rearrangement, bcl-2 is activated and B cells bearing the t(14;18) translocation express inappropriately elevated levels of the Bcl-2 protein. Figure 1. Schematic representation of the t(14; 18) translocation and its effects on B cells. Top. bcl-2 Bottom. Bcl-2 is a member of a larger family. Family members can interact with each other in a complex manner; some act to promote and others to inhibit apoptosis (14). The Bcl-2 protein protects cells from apoptosis, whereas its homologue, Bax, kills cells (21). Thus, the ratio of Bcl-2 to Bax is a determinant of susceptibility to apoptosis (14) (Figure 1, bottom). Strong expression of Bcl-2 protein has been observed in lymphoid infiltrates in liver and bone marrow specimens of patients with mixed cryoglobulinemia (22). In a previous study, the prevalence of bcl-2 rearrangement in peripheral blood mononuclear cells was significantly higher in patients with chronic HCV infection than in healthy persons or those without HCV infection but with chronic liver diseases or systemic autoimmune disorders (13). Of note, the prevalence of bcl-2 rearrangement was particularly high in patients with HCV-associated type II mixed cryoglobulinemia. We sought to evaluate the prevalence of bcl-2 rearrangement in peripheral blood cells of patients with mixed cryoglobulinemia, to confirm that results are patient specific by sequencing studies, to analyze Bcl-2 expression and the ratio of Bcl-2 to Bax in these patients, and to observe the effect of antiviral therapy. Methods Patients We enrolled 37 patients (12 men and 25 women; mean age SD, 64 9 years) with HCV infection and mixed cryoglobulinemia who were consecutively referred to the outpatient clinic of the Department of Internal Medicine, University of Florence School of Medicine, a tertiary hepatology center, and the rheumatologic section of the Department of Internal Medicine, University of Pisa School of Medicine, from January 1999 to May 2000. These patients were compared with 101 consecutively recruited patients (62 men and 39 women; mean age, 51 11 years) who had HCV-related chronic liver diseases but not mixed cryoglobulinemia or another lymphoproliferative disease. Hepatitis C virus infection was established by detection of circulating anti-HCV antibodies (EIA-2 and RIBA-2, Ortho Diagnostic Systems, Raritan, New Jersey) and HCV RNA (nested polymerase chain reaction [PCR] for HCV) (10, 13, 23). Essential mixed cryoglobulinemia was diagnosed according to published criteria (10, 13). Serum cryoglobulins, complement fraction levels, rheumatoid factor, and autoantibodies were routinely measured and characterized in all patients as described elsewhere (10, 13, 23). Diagnosis of liver disease was based on results of liver biopsy. Lymphomas were diagnosed by an independent pathologist and classified according to the revised European-American classification of lymphoid neoplasms (24). No patient tested positive for hepatitis B surface antigen, IgM anti-HBc, hepatitis B virus DNA, IgM anti-delta, antiEpsteinBarr virus, anti-cytomegalovirus, antiherpes simplex virus, or anti-HIV. No patient had a history of alcohol abuse or previous antiviral or immunosuppressive treatment. All patients gave informed consent to participate in the study, which was performed in accordance with the principles of the Declaration of Helsinki, and the study was approved by the local ethics committee. Detection of the t(14; 18) Translocation The t(14; 18) translocation in peripheral blood mononuclear cells was detected on total DNA by using nested PCR (major breakpoint region), as described elsewhere (13). Nested PCR is a variant of PCR; after an initial series of amplification cycles, templates are again amplified by using a second set of primers internal to the first ones. The resulting reaction is very specific and sensitive owing to specific binding to the target sequences of four instead of two specific primers. The limit of sensitivity was one rearranged cell in 105 to 106 normal cells. Amplification products were analyzed by both ethidium bromide staining and hybridization with a specific digoxigenin-labeled probe (Southern blot analysis). Each sample was analyzed at least twice, and all samples that tested negative on PCR were analyzed at least four times. Different cell samples that were obtained at the same time (synchronous) or at different times (metachronous) were also analyzed when possible. Approximately 2.5 105 mononuclear cells were tested in each reaction, corresponding to about 1 g of DNA. Positive and negative control samples were included in each experiment (13). To avoid false-positive results caused by carryover of PCR product, precautions were taken, as described elsewhere (10, 13). To ensure DNA amplificability, PCR was also performed by using primers for the human HLA gene (exon 2 of HLA-DRB gene), as previously reported (13). Finally, bcl-2/JH junction sequence was determined in part by cycle sequencing and solid-phase sequencing techniques (13, 25) and in part by automated sequencing (Abi Prism, Perkin Elmer, Norwalk, Connecticut). Measurement of Bcl-2 and Bax Proteins Bcl-2 and Bax proteins were measured as described elsewhere (13) on freshly isolated peripheral blood mononuclear cells and, when possible (9 patients), in separated cell subgroups (T cells, B cells, and monocytes and macrophages). Bcl-2 was detected by using monoclonal mouse anti-human Bcl-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, California), and Bax was detected by using polyclonal rabbit anti-human Bax (Upstate Biotechnology, Inc., Lake Placid, New York). The CD2+ T cells, CD19+ B cells, and CD14+monocytes and macrophages from peripheral blood were separated by immunomagnetic isolation using Dynabeads M450 Pan-T, M-450 Pan-B, and M-450 CD14+, respectively (Dynal A.S., Oslo, Norway), according to the manufacturers instructions. Statistical Analysis Data are expressed as the mean SD. Data were analyzed by performing the Fisher exact test, using True Epistat 4.0 statistical software (Epistat Service, Richardson, Texas). A P value less than 0.05 was considered significant. Role of the Funding Sources The funding sources had no role in the analysis, reporting, or interpretation of the data or in the decision to submit the report for publication. Results The Table shows the clinical, epidemiologic, and pathologic characteristics of patients with HCV-related mixed cryoglobulinemia. The mean duration of mixed cryoglobulinemia syndrome was 9.2 5.2 years. Most of these patients (91%) had chronic liver diseases. Liver biopsy showed chronic hepatitis in 27 patients (72.9%) and cirrhosis in 7 patients (18.9%); of the latter patients, 1 also had superimposed hepatocellular carcinoma. Liver biopsy was not performed in the remaining 3 patients becaus

Genetic determinants in hepatitis C virus–associated mixed cryoglobulinemia: Role of polymorphic variants of BAFF promoter and Fcγ receptors

OBJECTIVE Mixed cryoglobulinemia (MC) is a hepatitis C virus (HCV)-related immune complex disorder. Only some HCV-infected patients develop MC, which suggests that the genetic background of the host plays a key role. This study was undertaken to evaluate the contribution of host genetic factors in the pathogenesis of HCV-associated MC (HCV-MC) by analyzing allelic variants of low-affinity Fcγ receptor (FcγR) genes and BAFF promoter. METHODS FcγR polymorphisms (FCGR2A 131 R/H, FCGR2B 232 I/T, FCGR3A 176 V/F, and FCGR3B NA1/NA2) and BAFF promoter polymorphism -871 C/T were analyzed in 102 patients with HCV-MC and 108 patients with HCV without MC, using polymerase chain reaction-based techniques. RESULTS A higher prevalence of -871 T/T homozygosity (31% versus 16%; P = 0.001) and a greater frequency of T alleles of the BAFF promoter (80% versus 57%; P = 0.004) were found in the HCV-MC group than in the HCV group. A significant increase in serum BAFF concentration was significantly associated with the higher frequency of the T allele in HCV-MC (mean ± SD 4.12 ± 1.29 versus 2.09 ± 0.81 ng/ml; P < 0.0005). The distribution of the FcγR genotypes was not significantly different. In the 21 HCV-MC patients treated with rituximab, the response was strictly related to F allele homozygosity (significantly reduced in 5 of 5 patients with the FCGR3A F/F genotype versus 4 of 16 with V/V or V/F; P < 0.0005). CONCLUSION These results indicate the importance of host genetic background in the development of HCV-MC, suggesting that mechanisms enhancing Ig production and B cell survival may play a relevant role. Genetic FcγR variants seem to be crucial to the effectiveness of rituximab therapy.

Triple antiviral therapy in hepatitis C virus infection with or without mixed cryoglobulinaemia: a prospective, controlled pilot study.

BACKGROUND Mixed cryoglobulinaemia is strongly related to hepatitis C virus infection. Treatment with peg-interferon and ribavirin has been indicated as first-line therapy for mild/moderate hepatitis C virus-related mixed cryoglobulinaemia. AIM To evaluate the safety and efficacy of triple boceprevir-based antiviral therapy in patients with or without mixed cryoglobulinaemia previously treated with peg-interferon and ribavirin, and with advanced liver disease. METHODS Thirty-five hepatitis C virus-positive patients (17 with asymptomatic mixed cryoglobulinaemia, 5 with symptomatic mixed cryoglobulinaemia, and 11 without mixed cryoglobulinaemia) were treated with triple boceprevir-based antiviral therapy. RESULTS In 19/22 cryoglobulinaemic subjects (86%), the addition of boceprevir induced cryocrit disappearance. Cryocrit behaviour was related to virological response, with improvement of symptoms upon undetectable viraemia and reappearance after virological breakthrough. The rate of sustained virological response was lower in cryoglobulinaemic patients than in patients without mixed cryoglobulinaemia (23.8% vs 70% respectively, p=0.01). CONCLUSION Boceprevir-based therapy was safe and effective in cryoglobulinaemic patients. The correlation between direct inhibition of hepatitis C virus replication and clinical improvement in mixed cryoglobulinaemic patients is definitive proof of the key pathogenetic role played by viral replication. Further studies are needed to confirm and clarify the reduced virological response in patients with mixed cryoglobulinaemia.

Role of MicroRNA Profile Modifications in Hepatitis C Virus-Related Mixed Cryoglobulinemia

Hepatitis C virus infection is closely related to lymphoproliferative disorders (LPDs), including mixed cryoglobulinemia (MC) and some lymphomas. Modification of the expression of specific microRNAs (miRNAs) has been associated with different autoimmune diseases and/or LPDs. No data exist about the modifications in miRNA expression in HCV-associated LPDs. The aim of this study was to analyze the expression levels of a panel of miRNAs previously associated with autoimmune/LPDs in a large population of HCV patients with and without MC or non-Hodgkin’s lymphoma (NHL), to identify potential markers of evolution of HCV infection. PBMC expression of miR-Let-7d, miR-16, miR-21, miR-26b, miR-146a and miR-155 was evaluated by real-time PCR in 167 HCV patients (75 with MC [MC-HCV], 11 with HCV-associated NHL [NHL-HCV], 81 without LPD [HCV]) and in 35 healthy subjects (HS). A significant increase in miR-21 (p<0.001), miR-16 (p<0.01) and miR-155 (p<0.01) expression was detected in PBMCs from only NHL patients whereas a significant decrease in miR-26b was detected in both MC and NHL subjects (p<0.01) when compared to HS and HCV groups. A restoration of miR-26b levels was observed in the post-treatment PBMCs of 35 HCV-MC patients experiencing complete virological and clinical response following antiviral therapy. This study, for the first time, shows that specific microRNAs in PBMC from HCV patients who developed MC and/or NHL are modulated differently. The specific, reversible downregulation of miR-26b strongly suggests the key role it plays in the pathogenesis of HCV-related LPDs and its usefulness as a biomarker of the evolution of HCV infection to these disorders.

Association between persistent lymphatic infection by hepatitis C virus after antiviral treatment and mixed cryoglobulinemia.

To the editor: Hepatitis C virus (HCV) is closely related to the development of mixed cryoglobulinemia (MC). Occult HCV infection in sustained virologic responders (SVRs) after antiviral treatment has been shown,[1][1],[2][2] but MC patients have never been investigated. We studied 102 HCV

Can BAFF promoter polymorphism be a predisposing condition for HCV-related mixed cryoglobulinemia?

To the editor: Mixed cryoglobulinemia (MC) is a benign but prelymphomatous condition whose clinical manifestations are secondary to systemic immune complex–related vasculitis, which is the final step in a complex process initiated by unregulated B-cell expansion. MC is strongly associated with

Value of IL28B genotyping in patients with HCV‐related mixed cryoglobulinemia: results of a large, prospective study

HCV‐related mixed cryoglobulinemia (MC) is characterized by clonal expansion of B cells producing a polyreactive natural antibody (rheumatoid factor) and interferon (IFN)‐based therapy is the first therapeutic option in mild‐moderate MC. Single nucleotide polymorphisms (SNPs) proximal to genes involved in the innate response (IL28B/IFN‐λ gene family) are strongly associated with spontaneous and IFN‐induced viral clearance in hepatitis C, but no data exist about their role in HCV‐positive MC. A large cohort of patients with HCV and MC was studied to evaluate the influence of IL28B genotype on the response to treatment and/or the evolution to MC of HCV infection. The rs12979860/rs8099917 IL28B polymorphisms were analysed in 481 consecutive HCV‐positive subjects (250 with MC and 231 without MC, as controls) using real‐time PCR and direct sequencing. Hundred and fifteen HCV patients with MC received standard anti‐HCV therapy, and the results were evaluated according to the IL28B SNP distribution. Similar IL28B SNPs allele frequencies were recorded for patients and controls. IL28B major allele homozygosis (for both SNPs tested) was tightly correlated with virological and clinical response (P = 0.002). A statistically significant association was limited to ‘difficult‐to‐treat’ (G1/4) HCV genotypes. The IL28B genotype was a strong independent predictor of response (P = 0.007, OR 6.06; CI 1.65–22.22). The IL28B genotype was confirmed to be a useful predictor of response to IFN‐based therapy in patients with HCV and MC. The very close correlation between IL28B SNP distribution, virological and clinical response definitively confirmed the key role played by HCV in MC. Conversely, the IL28B genotype does not seem to influence the evolution to MC.

Hepatitis C virus core protein expression in human B-cell lines does not significantly modify main proliferative and apoptosis pathways.

Hepatitis C virus (HCV) chronic infection has been associated with many lymphoproliferative disorders. Several studies performed on hepatoma and fibroblast cell lines suggest a role of the HCV core protein in activation of cellular transduction pathways that lead to cell proliferation and inhibition of apoptosis. However, no data are available concerning the effects of HCV core expression on B-lymphocyte proliferation and apoptosis. B-lymphocyte cell lines permanently expressing full-length HCV 1b core sequences isolated from chronically infected patients were established using B-cell lines at different degrees of differentiation. Clones and pools of clones permanently expressing the HCV core were selected and characterized for protein expression by Western blot and FACS. Expression of HCV core proteins did not significantly enhance cell proliferation rates under normal culture conditions or under mitogenic stimulation. Analysis of NF-kappa B, CRE, TRE and SRE pathways by luciferase reporter genes did not show a significant influence of HCV core expression on these signal transduction cascades in B-lymphocytes. The effects of HCV core on anti-IgM and anti-FAS-induced apoptosis in B-cell lines was also analysed. In this experimental model, HCV core expression did not significantly modify the apoptotic profile of the B-lymphocyte cell lines tested. These data underline a cell type-specific effect of HCV core expression. In fact, it was not possible to show a significant contribution of the HCV core protein in activation of the major B-cell signal transduction pathways involved in the regulation of proliferation and programmed cell death, which is in contrast with the results reported in hepatoma cell lines.

Involvement of PI3K in HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) core protein has been shown to deregulate cell growth and programmed cell death in hepatoma cells, but only minimal informations are available about its possible role on B‐lymphoproliferative disorders (LPDs). The aim of our work was to analyze the biological activity of HCV core protein on B‐cell proliferation. We established Wil2‐ns and Ramos B‐cell lines that stably expressed the HCV core protein. Growth curve, thymidine incorporation analysis, as well as the expression of PCNA and activated‐ERKs demonstrated that HCV core protein induced an increased growth in both cell lines. Interestingly, the HCV core protein expression determined, in our model, a downregulation of DNp73 and an upregulation of DNp63, which was essential for the maintenance of viral‐dependent effects on cell growth. Finally, we have identified phosphoinositide 3‐kinase (PI3K) as mediator of HCV core‐dependent transcriptional increase of DNp63, which in turn correlated with the increasing of lymphocyte proliferation. In primary B‐lymphocytes, derived from HCV‐related low‐grade non‐Hodgkins lymphoma patients, consistent results were obtained. These findings provide evidence for a possible pathogenetic role played by HCV core protein in HCV‐related lymphomagenesis; it could occur through the deregulation of PI3K activity, consequent activation of Akt and overexpression of DNp63. J. Cell. Physiol. 214: 396–404, 2008.

Improvement in liver cirrhosis after treatment of HCV-related mixed cryoglobulinemia with rituximab

Mixed cryoglobulinemia (MC) is the most strictly virus-related extrahepatic HCV disease. Antiviral therapy is considered the first therapeutic option; however, MC patients are frequently excluded from treatment due to contraindications. The effectiveness of B-cell depletion by anti-CD20 monoclonal antibody (rituximab) has recently been described, but the possibility of an immunodepression- related increase in viral replication and aminotransferase values limits its use in patients with advanced liver disease. Unfortunately, MC patients frequently also have cirrhosis. To our knowledge, no data are available regarding the effect of rituximab therapy in patients with decompensated cirrhosis. We report the successful treatment with rituximab (4 weekly infusions of 375 mg/m 2) of two patients (a 58-year-old man, and a 65-year-old woman) with HCV-related MC syndrome and decompensated liver cirrhosis. These patients underwent at least 6 months of post-treatment follow-up. In both cases a consistent improvement of MC syndrome was evident after treatment. In addition, improvement of liver protidosynthetic activity, increased prothrombin time, impressive reduction or disappearance of ascites and encephalopathy were also observed, in spite of some increase in viral titers or in ALT values. The Child-Pugh score improved from B8 to A6 and from Cll to B7, respectively. Pre- and post-treatment transjugular liver biopsies were available in 1 patient, showing disappearance of lymphocytic infiltration after treatment. These case reports show the effectiveness and safety of rituximab in patients with HCV-related MC and advanced cirrhosis, and strongly suggest that the depletion of CD20+ B-cells induced by rituximab treatment may be responsible for liver function improvement. The mechanisms involved are unknown. Interesting working hypotheses may implicate a role played by B-cell infiltrates in conditioning liver damage. The improvement of Kupffer cell function due to the cryocrit value reduction might also play a role.