Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation

Rheumatology 2007;46:1234 1242 Advance Access publication 12 June 2007 doi:10.1093/rheumatology/kem132 Review Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation D. Saadoun 1,2, D. A. Landau 1,2, L. H. Calabrese 3 and P. P. Cacoub 1,2 Hepatitis C virus (HCV) infection is the second most cocmmon chronic viral infection in the world with a global prevalence of about 2%. Chronic HCV infection is commonly associated with a number of extrahepatic complications. Circulating mixed cryoglobulins (MCs) are detected in 40 60% of HCV-infected patients whereas overt cryoglobulinaemia vasculitis develops in only 5 10% of the cases. MC reflects the expansion of B cells producing a pathogenic IgM with rheumatoid factor (RF) activity. Because cryoglobulin-producing B cells in HCV are mostly monoclonal, HCV-associated MC can be viewed as a benign B cell lymphoproliferative condition. The disease expression of MC vasculitis is variable, ranging from mild clinical symptoms (purpura, arthralgia) to fulminant life-threatening complications (glomerulonephritis, widespread vasculitis). The overall risk of non-hodgkin s lymphoma in patients with HCV-MC is estimated to be 35 times higher than that in the general population. This review will focus on recent advances in our understanding of the clinical course, complications, pathophysiology and treatment of those immune-mediated disorders. KEY WORDS: HCV infection, Mixed cryoglobulinaemia, Vasculitis, Non-Hodgkin s lymphoma. Introduction Hepatitis C virus (HCV) infection is the second most common chronic viral infection in the world, with over 170 million cases worldwide and a global prevalence of about 2% [1]. HCV is an RNA virus belonging to the Flaviviridae family. HCV infection is characterized by a high rate of chronicity leading to chronic liver inflammation resulting in cirrhosis and/or hepatocellular carcinoma in 20%. HCV is uniquely associated not only with chronic hepatic inflammation but also an array of extrahepatic complications. In the majority of these associated extrahepatic manifestations the pathogenic mechanism appears to be immunologically driven, with features of autoimmunity in many. Table 1 represents a partial list of immunological laboratory abnormalities and disorders that have been associated with HCV infection. In terms of the strength of association of these disorders, several have strong links based on epidemiological and clinical studies while for others the supporting data are limited or conflicting. Among these extrahepatic manifestations, cryoglobulinaemia and its clinical sequelae hold the strongest association. The history of cryoglobulinaemia as a disease dates back to the 1930s when Wintrobe and Buell [2] described its association in a single patient with multiple myeloma. The modern era of cryoglobulinaemia is dated to 1966 when Meltzer and colleagues [3] described nine patients with cryoglobulins but lacking any known disease process associated with such cryoproteins. Among these patients were eight women and one man with a characteristic clinical syndrome characterized by purpura, arthralgia and weakness. This seminal article on the disorder recognized the importance of hepatic involvement but these patients had no form of infective hepatitis recognized at the time. Over the ensuing years several groups suggested hepatitis B as the etiological agent responsible for cryoglobulinaemia [4, 5] 1 Université Pierre et Marie Curie-Paris 6, CNRS, UMR 7087, 2 AP-HP, Hôpital Pitié-Salpêtrière, Service de Médecine Interne, Paris, F-75013 France and 3 Department of Rheumatic and Immunologic Diseases, Cleveland Clinic Lerner, Cleveland, OH, USA. Submitted 7 January 2007; revised version accepted 11 April 2007. Correspondence to: Prof. Patrice Cacoub, MD, PhD, AP-HP, Hôpital Pitié-Salpêtrière, Service de Médecine Interne, Paris, F-75013 France. E-mail: patrice.cacoub@psl.aphp.fr though others failed to confirm these results [6, 7]. In 1989, Houghton and colleagues [8] using molecular techniques cloned an RNA molecule with the characteristic of a small single-stranded RNA virus and named it HCV. Soon after Pascual and colleagues [9] noted the strong association between HCV and what was previously labelled as essential cryoglobulinaemia and since then this association has been extensively reported with up to 80 90% of the patients with cryoglobulinaemic vasculitis chronically infected with HCV. This review will focus on recent advances in our understanding of the clinical course, complications, pathophysiology and treatment of those immune-mediated disorders most strongly linked to HCV and cryoglobulinaemia. HCV-associated cryoglobulinemia Laboratory features Cryoglobulins are defined by the presence of circulating immunoglobulins that precipitate as serum is cooled below core body temperature and resolubilize when rewarmed. Cryoglobulins are TABLE 1. Extrahepatic immunological complications associated with chronic HCV infection Established associations Mixed cryoglobulinaemia Membranoproliferative glomerulonephritis Porphyria cutanea tarda Sialadenitis Autoantibody production (RF, ANA, SMA, ATG, ACL) Possible associations B-cell non-hodgkin s lymphomas Arthralgia, myalgia Diabetes melitus Polyarteritis nodosa Autoimmune cytopenia Autoimmune thyroiditis Interferon-induced Dysthyroidism Psoriasis Lichen planus Sarcoidosis Polyneuropathy Cutaneous vasculitis RF, rheumatoid factor; ANA, antinuclear antibodies; SMA, anti-smooth muscle antibodies; ATG, antithyroglobulin antibodies; ACL, anticardiolipin antibodies. 1234 ß The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

HCV-related MC 1235 readily detectable in 40 60% of HCV-infected patients in most case series [10 13]. Cryoglobulins are immunochemically categorized into three types by the method of Brouet et al. [14]. Type I cryoglobulins are single monoclonal immunoglobulins usually found in haematological diseases. Type II consists of polyclonal IgG with monoclonal rheumatoid factor (RF) activity. Types III are comprised of polyclonal IgG and polyclonal RF. Types II and III are often referred to as mixed cryoglobulins (MCs) [14]. Beside the detection of serum cryoglobulin itself, other laboratory abnormalities may provide surrogate evidence of the presence of cryoglobulinaemia such as low C4, depressed total haemolytic complement levels, monoclonal proteinaemia or RF activity. Most HCV-MC vasculitis patients demonstrate decreased levels of the early complement components C1, C4 and C2, whereas C3 levels fluctuate with the disease course. An RF activity is frequently observed, in up to 70% of the patients (Table 2). Electrophoresis and immuno-electrophoresis may show polyclonal hypergammaglobulinaemia or monoclonal Ig (mostly IgMk). Other immunological abnormalities in HCV-infected patients, whether MC-positive or -negative, include antinuclear (17 41%), anti-cardiolipin (20 27%), anti-smooth muscle cell (9 40%) and anti-thyroglobulin antibodies (8 13%) with important geographic differences in the prevalence [15 18]. There is usually no correlation between non-organ-specific autoantibodies and the main HCV-related features [18]. Main clinical features Circulating MCs are frequently detected in HCV-infected patients (40 60%) whereas overt cryoglobulinaemia vasculitis develops in only 5 10% of the cases [10]. The most frequent target organs are skin, joints, nerves and kidney. The disease expression is variable, ranging from mild clinical symptoms (purpura, arthralgia) to fulminant life-threatening complications (glomerulonephritis, widespread vasculitis). The most common clinical and immunological manifestations of HCV-associated MC are shown in Table 2. Purpura. Skin is the most frequently involved target organ and is the direct consequence of the small-size vessel vasculitis. The main sign is palpable purpura which is reported in 70 90% of the patients [19, 20]. It always begins at the lower limbs and may extend to the abdominal area and less frequently to the trunk and upper limbs. With petechial or papular, seldom necrotic aspect, this purpura can be compounded of erythematosus maculae and dermal nodules. It persists 3 10 days with a residual brownish pigmentation. Skin biopsy shows a non-specific leucocytoclastic vasculitis involving small-size vessels with inflammatory infiltrates and, in some cases, fibrinoid necrosis of the arteriolar walls and endovascular thrombi. The association with other dermatological symptoms, like upper malleolar ulcers and urticarian vasculitis TABLE 2. Main features in hepatitis C-associated MC a Trejo et al. [150] Ferri et al. [63] Saadoun et al. [151] Findings n ¼ 206 n ¼ 231 n ¼ 118 Female/male ratio 1.5 3.0 1.1 Age at diagnosis (years) 54 56 67 Purpura 67 (32) 187 (81) 75 (64) Arthralgia 82 (40) 166 (72) 68 (57) Peripheral neuropathy 35 (17) 134 (58) 51 (43) Sicca syndrome 40 (19) 65 (29) 21 (18) Renal involvement 115 (56) 46 (20) 16 (14) Raynaud s phenomenon 23 (11) 79 (36) 5 (4) Leg ulcers 9 (4) 25 (11) 2 (2) Rheumatoid factor 107 (52) 231 (100) 93 (79) Type II cryoglobulin 143 (62) 100 (84) Low C4 level 98 (56) 64 (54) ANA 92 (52) 21 (18) a Except where indicated otherwise, values are expressed as n (%). ANA, antinuclear antibodies. is not rare. Raynaud s syndrome and acrocyanosis, which may evolve to digital ulcerations, can also occur. Arthralgia. Arthralgia is reported in 40 80% of HCV-infected patients with MC [21, 22]. They are bilateral and symmetric, non-deforming and touched mainly great articulations, knees and hands, more seldom elbows and ankles. Frank arthritis is rarely reported, being present in <10% of the patients [21, 22]. RF activity is found in 70 80% of the MC patients but is not correlated with the presence of articular disease as patients chronically infected with HCV in the absence of HCV-MC or RF may have prominent articular symptoms. There is no evidence of joint destruction, and antibodies to cyclic citrullinated peptide, which are highly specific of rheumatoid arthritis, are absent [23, 24]. Neuropathy. The prevalence of peripheral nervous system involvement varies in the literature and can be as high as 55% of the cases [25]. Neurological manifestations range from pure sensory axonopathy to mononeuritis multiplex. The most frequently described form is a distal sensory or sensory-motor polyneuropathy (PN). Bilateral, more often asymmetrical polyneuropathies represent 45 70% of the MC-PN and mononeuropathies multiplex 30 55% [26]. Motor deficit is inconsistent and mainly affects the lower limbs, appearing a few months to a few years after sensory symptoms. The tempo of the vasculitic neuropathy may be subacute, chronic or acute on chronic. In patients with distal polyneuropathy, nerve conduction studies are in keeping with a predominantly axonal process, mainly affecting sensory nerves. Neuropathological data show axonal degeneration, differential fascicular loss of axons, signs of demyelinization and small-vessel vasculitis with mononuclear cell infiltrates in the perivascular area [25]. Reports on well-documented central nervous system (CNS) involvement in patients with HCV-associated vasculitis are rare [25, 27 29], though it has been described as the initial extra hepatic manifestation of HCV infection. Clinically, transient ischaemic attacks, stroke, progressive reversible ischaemic neurological deficits, lacunar infarctions or encephalopathic syndrome may occur [25, 27]. MRI findings of the brain have been consistent with ischaemia, showing either small lesions of the periventricular white matter and the cerebral trunk or extensive supra- and infratentorial white matter lesions suggesting cerebral vasculitis [25]. The presence of CNS vasculitis involving small- and medium-sized vessels has been documented in two patients [30, 31]. Renal involvement. Renal manifestations are reported in 20 35% of the MC patients [19, 32, 33]. The most frequent clinical and histological picture is that of acute or chronic type-i membranoproliferative glomerulonephritis (MPGN) with sub-endothelial deposits [34]. It represents >70 80% of cryoglobulinaemic renal diseases and it is strongly associated with the type II cryoglobulinaemia with IgM RF [34 36]. The most frequent presentation (55%) is proteinuria with microscopic haematuria and a variable degree of renal insufficiency [37]. Acute nephrotic (20%) or acute nephritic syndrome (25%) can also reveal cryoglobulinaemic renal involvement [35]. New onset arterial hypertension is seen in 80% of the cases. Early complement components (C1q, C4) are usually very low. Chronic uraemia may develop in 10 20% of the MC patients but only after prolonged follow-up of a decade or more [35]. Morphological features are characterized by important monocytes infiltrates with double contours of the basement membrane, large, eosinophilic and amorphous intra-luminal thrombi. In indirect immunofluorescence, intra-glomerular sub-endothelial deposits of IgG, IgM and complement components are observed. The electron microscopic features with sub-endothelial and intra-luminal deposits presenting a crystalloid aspect are pathognomonic. Vasculitis of small renal arteries or extra-capillary crescents are rarely observed [33 35, 38]. A number of

1236 D. Saadoun et al. less common nephropathies have been reported, including mesangioproliferative or focal segmental glomerulonephritis. Liver involvement. Clinical and biological evidence of liver disease is evidenced in 60 80% of the patients with essential MC whose liver biopsies showed chronic active hepatitis or cirrhosis [5, 39]. In asymptomatic HCV-infected patients, the significance of cryoglobulins in the development of cirrhosis has been the subject of speculation. A meta-analysis of 19 studies on a total of 2323 patients with chronic HCV infection found a 44% prevalence of cryoglobulinaemia and a highly significant association between cirrhosis and cryoglobulinaemia after adjustment for age, gender and estimated duration of disease [40]. In a recent study of 437 consecutive HCV-infected patients [41], advanced liver fibrosis and steatosis were found in 37 and 35% of MC patients, respectively. In multivariate analysis, MC increased by nearly 3-fold the risk of having advanced liver fibrosis and steatosis. Other manifestations. Other organs may more rarely be involved. Abdominal pains are reported and gastrointestinal bleeding secondary to mesenteric vasculitis has been described [22, 42]. Lungs can be involved more frequently without clinical symptoms but some patients may present moderate effort dyspnoea, dry cough, interstitial lung fibrosis, pleural effusions or haemoptysis, which can be consequent of intra-alveolar haemorrhages [43, 44]. Cardiac involvement including mitral valvular damage, coronary vasculitis complicated by myocardial infarction, pericarditis or congestive cardiac failure all have been described [45]. HCV-MC can also rarely manifest as chronic fever of unknown origin. Sicca syndrome and Sjo gren s syndrome. Other autoimmune conditions associated with HCV have also been reported [46 48]. Studies have suggested an epidemiological association between Sjo gren s syndrome and HCV infection [49, 50]. HCV seropositivity was found in 156 (12%) of the 1309 reported SS patients tested for HCV infection [50]. Symptomatic xerophthalmia and xerostomia affects 10 53% of HCV-infected patients [49, 51], although this association was not demonstrated in a large cohort of US veterans [52]. However, this latter study only included males, a bias that greatly underestimated the prevalence of sicca syndrome [52]. HCV treatment was not demonstrated as effective for sicca syndrome by Cacoub et al. [53] whereas Doffoel-Hantz et al. [54] found improvement of sicca features in one-third of the SS patients treated with anti-hcv therapy. The main differential aspect between primary and HCV-related Sjo gren s syndrome is the immunological pattern, with a predominance of MC-related markers (low C4, RF and MC) in HCV-related Sjo gren s syndrome [46]. A sicca syndrome has been reported in 20 40% of MC patients, however, those meeting most definitions of definite Sjo gren s syndrome are less commonly encountered. Some authors have distinguished the sicca syndrome of HCV infection from Sjo gren s syndrome based on several differences, including absence of anti-ssa and anti-ssb antibodies, pericapillary and non pericanalary, lymphocytic infiltrate, and lack of glandular canals damages [49]. However, others studies showed that HCV-related sialadenitis was indistinguishable from those patients with primary Sjo gren s syndrome [15, 55]. In a large series of HCV-related SS patients, the prevalence of anti-ssa and anti-ssb antibodies was nearly 20% [55]. The mechanisms through which HCV may induce sialadenitis include its sialotropism [51]. In rodent models, the expression of the HCV E1 and E2 glycoproteins by transgenic mice is associated with the development of sialadenitis [56]. Natural history of cryoglobulinaemic vasculitis During chronic HCV infection, the immunochemical properties of serum cryoglobulins may fluctuate. The detection of type II mixed cryoglobulins is more stable over time than type III and oligoclonal MC [57]. The oligoclonal type appears to be an intermediate stage in the course of type III changing to type II MC. Circumstances predisposing HCV-infected patients to develop cryoglobulinaemic vasculitis remains unclear. Interaction between HCV and lymphocytes directly modulates B- and T-cell function [58] and result in polyclonal activation and expansion of B cell producing IgM with RF activity. CD4þCD25þ regulatory T cell, which have been shown to control autoimmunity, are significantly reduced in HCV-MC vasculitis patients [59]. This defect in immune regulation may account for the expansion of peripheral autoreactive B cell that drive MC vasculitis. Furthermore, HLA type II polymorphism may predispose to HCV-MC. HLA-DR11 is associated with MC vasculitis whereas HLA-DR7 appears to protect from the production of type II MC [60]. In contrast, specific virological factors have not yet been identified. Specific changes in HCV envelope sequence distribution are unlikely to be directly involved in the establishment of pathological B-cell monoclonal proliferation [61]. Cryoglobulinaemic vasculitis is usually associated with advanced age, longer duration of HCV infection, type II MC, a higher MC serum level and clonal B-cell expansions in both the blood and liver [57, 62]. The natural history and prognosis of MC vasculitis are variable and highly dependent on renal involvement or on the extent of vasculitis lesions. The worse pronostic factors are an age older than 60 yrs at diagnosis and renal involvement [63]. The overall 5 yrs survival after the diagnosis of vasculitis range from 90% to 50% in case of renal involvement [37]. In historic series by Meltzer et al. [3] and Gorevic et al. [30], renal involvement was the main cause of death. Even in the absence of significant renal failure, increased mortality from liver involvement, cardiovascular disease, infection and lymphoma has been reported [63]. In a recent retrospective Italian study of 231 patients, 79 of 97 deaths were linked to vasculitis (46%, of whom one-third due to renal involvement), cancer or haemopathy (23%), or liver disease (13%) [63]. Life-threatening MC complications are observed in up to 10% of the patients with almost two-thirds of death [64]. Intestinal ischaemia, pulmonary haemorrhage, high cryocrit levels and type II MC are associated with severe prognosis [64]. HCV and B cell non-hodgkin s lymphoma (NHL) Clinical aspects The lymphomagenic role of HCV is still a matter of debate. In HCV-infected patients, lymphoproliferative processes may evolve out of MC type II [65], or independently in patients without MC [66]. In long-term follow-up studies, only 4 6% of the patients with HCV-type II cryoglobulinaemia develop frank B-cell malignancies [67, 68], although in a recent multicentre Italian study, the estimated risk for lymphoproliferative disease was found to be 35-fold in patients with MC compared with the general population [69]. Epidemiological studies on the prevalence of HCV infection in B-cell NHL patients have yielded conflicting results [70 72]. Data from several countries indicate that the prevalence of HCV infection among patients with B-cell NHL ranges from 9 to 37%, values that are significantly higher than those found in patients with other haematological disorders, or in the general public [70, 73 78]. In contrast, several reports have not found an increased prevalence of HCV infection among patients with B-cell NHL [79, 80], suggesting a geographical influence that may signify that other factors are involved [72, 81, 82]. Meta-analysis studies have also investigated this association. In a meta-analysis published in 2003,

HCV-related MC 1237 Gisbert et al. [83] have shown that the prevalence of HCV infection in patients with B-cell NHL is 15%. An additional meta-analysis, published in 2004 by Matsuo et al. [84], estimated the odds ratio (OR) for NHL for HCV seropositive relative to seronegative persons at 5.7 [95% confidence interval (CI) 4.09 7.96]. This meta-analysis also included a comparison of studies performed in endemic vs non-endemic countries which showed similar results for both. Additionally, studies that have utilized blood donors as a control group had higher OR compared with studies that have chosen different control groups (OR ¼ 8.43 and 4.65, respectively). Certain histological subtypes of lymphomas may be more strongly associated with HCV infection. Generally, low-grade lymphomas are more frequently associated with HCV than high-grade lymphomas [82]. In most studies, marginal zone B-cell lymphoma (MZL), lymphoplasmacytic lymphoma/immunocytoma and diffuse large B-cell lymphoma are the more frequent histological subtypes reported in HCVinfected patients [66, 74, 85 87]. A large case series published by Trejo et al. [88] has also demonstrated a frequent extra-nodal localization of NHL in HCV-infected patients and in particular, the liver and the major salivary gland were significantly overrepresented. An association between HCV infection and other B-cell lymphoproliferative processes, such as chronic lymphocytic leukaemia and multiple myeloma, has been suggested but was not demonstrated in a consistent manner, and thus remains debatable [82]. Pathogenesis Direct lymphocyte transformation by a given microbial agent account for lymphotropic transforming viruses such as Epstein Barr virus, human herpesvirus 8 and human T lymphotropic virus 1 that directly infect a subset of lymphoid cells in which they express viral oncogenes. The mechanisms by which HCV leads to the development of B-cell lymphoma remain to be elucidated. HCV may also exert a direct oncogenic activity on B cells. There is evidence at least in some experimental conditions for a direct effect of some HCV proteins, such as HCV core and NS3 on cellular proliferation and viabilty [89, 90]. HCV is capable of infecting B lymphocytes through LDL receptors or CD81 [91, 92]. HCV is a positive, single-strand RNA virus but without a DNA intermediate in its replicative cycle ruling out the possibility of an insertional oncogenesis. Although HCV can infect B cells in vitro, only one case of B-cell lymphoma associated with direct infection of B cells by HCV has been described so far [93]. Recently, active replication of HCV was demonstrated in cells (mostly CD20 B cells) of peri-hepatic lymph nodes of TABLE 3. Efficacy of antiviral treatment in B-cell Non-Hodgkin s Lymphoma associated with Hepatitis C infection cirrhotic patients [94]. Alternatively, HCV can exert its oncogenic potential indirectly by interacting with the host immune system [95]. HCV could exert a chronic stimulus on the immune system, leading to the selection of abnormal clones, in a scenario reminiscent of the role of Helicobacter pylori in gastric mucosalassociated lymphoid tissue (MALT) lymphoma [96]. In MC patients, the BCR was demonstrated to bind with HCV-E2 and NS3 [97, 98]. Clonal B-cell expansion has been demonstrated in liver, blood and bone marrow of patients with chronic HCV infection in the absence of overt B-cell malignancy [62, 99]. The B-cell repertoire in cryoglobulinaemic vasculitis is highly restricted. B cell clones in HCV-associated MC and lymphomas often use the VH1-69 gene and VkA27 segment which is also used by anti-e2 antibodies elicited by HCV [100, 101]. Sequencing of these Ig variable regions has also revealed that they are the product of somatic hypermutation [102, 103]. Data support the hypothesis that MC and the subsequent evolution to NHL are antigen-driven processes possibly sustained by HCV [104]. The increased percentage of extra-nodal marginal cell lymphomas in HCV infection and the report of a more than 10-fold greater risk for developing B-NHL of the liver and salivary gland than the risk of lymphomas at other sites is consistent with the hypothesis that B-NHL arise selectively from marginal zone B cell [105]. Antigenic stimulation of spleen marginal zone B cells by persisting HCV antigens and particularly the E2 viral protein might be involved in the pathogenesis [97, 106]. In this line, enhanced mutations of immunoglobulin heavy chain and proto-oncogenes (p53, Bcl2 and -catenin) have been demonstrated in HCV-associated lymphoma [107]. A higher rate of bcl-2 over-expression was reported in HCV patients with MC compared with HCV-infected patients without MC, with a further increase in patients with MC type II and NHL [108, 109]. Although HCV-associated bcl-2 over-expression in the mrf producing B cells (i.e. in cryoglobulins producing B-cell clones) remains to be convincingly demonstrated [110], the strongest argument for a role of HCV infection in NHL is provided by interventional studies (Table 3). A recent systematic review [111] summarized the response rate of lymphoproliferative disorders to anti-viral treatment in 65 HCV-infected patients, in a total of 16 different reports [93, 109, 112 125]. Complete remission was achieved in 75% (95% CI: 64 84%) of the cases. Additional studies published after the systematic review supported these results [126, 127]. Therapy for cryoglobulinaemic vasculitis With the discovery of HCV as the aetiological agent for most cases of mixed cryoglobulinaemia, new opportunities and Histological subtype n Antiviral treatment SVR (%) Haematological response (%) CR PR NR Relapse SLVL[122, 125, 126] 20 IFN (n ¼ 8) 6 (75) 7(87) 1(13) 0 1(13) IFNþRIBA (n ¼ 11) 8 (72.7) 7 (63.6) 4(36.3) 0 1(9) PegIFNþRIBA (n ¼ 1) 1(100) 1(100) 0 0 0 MZL [125, 127, 152, 153] 18 IFN (n ¼ 1) 1(100) 0 0 1(100) 0 IFNþRIBA (n ¼ 4) NA 2(50) 1(25) 1(25) 0 PegIFNþRIBA (n ¼ 13) 6 (50) b 9 (69.2) 3(23) a 1 (11.1) 0 Immunocytoma [112, 121, 127] 10 IFN (n ¼ 7) 4(57.1) 4(57.1) 0 3(42.8) 0 PegIFNþRIBA (n ¼ 3) 2 (66) b 1 (33.3) 2 (66.6) a 0 0 Follicular [127] 1 PegIFNþRIBA (n ¼ 1) 1(100) 1 (100) 0 0 0 Large B cell [154] 1 IFN (n ¼ 1) 1 (100) 1 (100) 0 0 0 Mantle cell [93] 1 PegIFNþRIBA (n ¼ 1) 1(100) 1(100) 0 0 0 Lymphocytic [142] 1 IFNþRIBA (n ¼ 1) 0 0 0 1(100) 0 Multiple myeloma [124] 1 IFN (n ¼ 1) NA 0 1(100) 0 0 MALT [116] 1 IFN (n ¼ 1) NA 0 1(100) 0 0 a Including one patient with stable disease, b Results for one patient not available. SLVL, splenic lymphoma with villous lymphocytes; MZL, marginal zone lymphoma; INF, interferon; RIBA, Ribavirin; PEG INF, pegylated interferon; SVR, sustained viral response; CR, complete remission; PR, partial remission; NR, no response; NA, not available.

1238 D. Saadoun et al. problems for crafting therapy of HCV-MC have emerged. A new and major concern was the potential adverse effects that immunosuppressive therapy with glucocorticoids and cytotoxic drugs could have on an underlying chronic viral infection. Alternatively, the discovery of HCV provided the opportunity to control HCV-MC with antiviral therapy based on the belief that the underlying infection was driving immune complex formation and resultant vasculitis. Unfortunately, the initial efforts to treat HCV with weekly interferon were associated with low rate of virological cure. Furthermore, the cornerstone of HCV antiviral therapy has been and continues to be interferon which has the potential to excacerbate autoimmune disease states [128]. Though rare, interferon has been documented to induce vasculitis in some patients previously without it and to exacerbate certain complications such as ulcer healing or neuropathy thus confounding the use of interferon-based therapies as sole treatment of HCV-MC [129]. While optimum therapy is still unclear a series of recent studies suggest a role for both antiviral as well as limited immunosuppressive therapy in the treatment of HCV-MC depending on the activity and severity of the underlying vasculitis and the status of the underlying infection. Figure 1 represents the therapeutic options in patients with HCV-MC. Antiviral agents The treatment of HCV infection (i.e. in the absence of HCV-MC) has progressed dramatically over the past 15 years with now the standard of pegylated interferon- and ribavirin therapy leading to sustained virological clearance in nearly two-thirds of the patients. The early attempts to control HCV-MC with standard thrice weekly IFN- was not surprisingly associated with a relatively poor response and a high relapse rate, especially in severe cases [130]. IFN- monotherapy was effective in 50 100% of the patients with purpuric skin lesions, but did not clearly demonstrate efficacy on neurological or renal involvement. However, when follow-up was sufficient, most of the responders developed virological and clinical relapses following IFN- withdrawal [130 132]. Combination therapy with standard IFN- plus ribavirin has provided much better short- and long-term results in patients with HCV-related vasculitis than historically reported with IFN-. In three uncontrolled studies [133 135], combination therapy with standard IFN- and ribavirin demonstrates enhanced efficacy on main HCV-related vasculitic manifestations (cutaneous, 100%; renal, 50% and neural, 25 75%). Two studies reported a loss of proteinuria and haematuria in sustained viral responders treated by IFN- FIG. 1. Therapeutic options in patients with HCV-associated MC vasculitis. Hepatitis C virus (HCV) binds to CD81 on the surface of B cells. This interaction enhances signalling through the BCR and may promote B-cell proliferation and clonal expansion. The harmful role of cryoglobulins is clear from the presence of Ig molecules and complement fractions in the wall of microvessels. A leucocytoclastic reaction may play a role in vessel damage. T cells and macrophages are the dominant infiltrating cells in vascular walls, and a T cell-mediated process appears to be the primary mechanism of vessel injury. The main targets organs in MC vasculitis are the skin (purpura and/or leg ulcer), the nerve (peripheral neuropathy) and the kidney (membranoproliferative glomerulonephritis). Antiviral therapy may clear the antigen (i.e. HCV) trigger of MC vasculitis. Anti-CD20 mab target the B-cell, involve in the cryoglobulin formation and possibly in the antigen presentation to T lymphocytes. Immunosuppressive agents and corticosteroids control the cellular and humoral immune response responsible for vasculitic lesions. Plasmapheresis may be useful for patients presenting with the most fulminant presentations including peripheral necrosis of extremities or rapidly progressive nephritis. APC, antigen presenting cell; M, macrophage; mab, monoclonal antibody.

HCV-related MC 1239 plus ribavirin [136, 137]. We recently reported in 72 consecutive HCV-MC patients that Peg-IFN- plus ribavirin achieved a higher rate of complete clinical (67.5 vs 56.2%), virological (62.5 vs 53.1%) and immunological response (57.5 vs 31.2%) as compared with standard IFN- plus ribavirin, regardless of HCV genotype and viral load [138]. In multivariate analysis, an early virological response (OR, 3.53; 95% CI 1.18 10.59) was independently associated with a complete clinical response of MC. A glomerular filtration rate <70 ml/min (OR 0.18; 95% CI 0.05 0.67) was negatively associated with a complete clinical response of MC [138]. Although, HCV-MC patients with HCV genotype 1 and/or previous failure to therapy displayed a lower clinical response rate, these factors were not independently associated with a poor clinical response in multivariate analysis [138]. Epidemiological features, HCV viral load, transaminases or liver damage did not influence the clinical outcome in this study [138]. The sub-group analysis of the 40 HCV-MC patients treated with Peg-IFN- plus ribavirin showed a complete recovery of skin involvement in 21/24 (87.5%), arthralgia in 18/22 (81.8%), peripheral neuropathy in 20/27 (74%) and nephropathy in 5/10 (50%) cases, respectively [138]. Immunosuppressive agents Immunosuppressive agents are typically reserved for patients with severe disease manifestations such as membranoproliferative glomerulonephritis, severe neuropathy and life-threatening complication. Traditionally a combination of corticosteroids and immunosuppressants such as cyclophosphamide and azathioprine have been used for the control of severe vasculitis lesions while awaiting the generally slow response to antiviral treatments. Mycophenolate mofetil may represent an alternative therapeutic option in patients refractory or intolerant to these immunosuppressive drugs [139]. In a large retrospective study of 105 patients with renal disease associated with cryoglobulinaemia vasculitis, 80% were administered corticosteroids and/or cytotoxic agents, while 67% underwent plasmapheresis [37]. Despite this aggressive approach, long-lasting remission of the renal disease was achieved in only 14% of the cases, and the 10-yr survival rate was only 49%. Corticosteroids, used alone or in addition to IFN-, did not favourably affect the response of HCV-related vasculitic manifestations in two controlled studies [131, 132]. In one randomized trial, methylprednisolone (MP) alone given for 1 yr was associated Therapeutic Strategies in HCV-related Cryoglobulinaemic Vasculitis with clinical response in 22% of the patients, compared with 66 and 71% in patients receiving IFN- or IFN- plus MP, respectively [132]. Low-dose corticosteroids may help to control minor intermittent inflammatory signs such arthralgia, but do not succeed in cases of major organ involvement (i.e. neurological, renal), or in the long-term control of vasculitis. Plasmapheresis offers the theoretical advantage of removing the pathogenic cryoglobulins from the circulation of patients with HCV-MC vasculitis. Immunosuppressive therapy is usually associated with plasma exchange in order to avoid the rebound increase in cryoglobulinaemia that is commonly seen after discontinuation of apheresis [35]. When used in combination with anti-hcv treatment, plasmapheresis did not modify the virological response if IFN- was given after each plasma exchange session [140]. Recently, several groups have reported on the efficacy of anti-cd20 monoclonal antibody (rituximab) in patients with HCV-MC vasculitis [141 148]. Such an approach involves the use of monoclonal antibodies directed to CD20 antigen, a transmembrane protein expressed on pre-b lymphocytes and mature lymphocytes. Rituximab proved effective on skin vasculitis manifestations, subjective symptoms of peripheral neuropathy, arthralgia and low-grade B-cell lymphoma. The pooled data of the eight rituximab studies including 43 HCV-MC patients showed a complete recovery of skin involvement in 24/33 (73%), arthralgia in 16/30 (53%), peripheral neuropathy in 9/25 (36%) and nephropathy in 9/13 (70%) cases, respectively [141 148]. Most clinical responders also had a decrease in serum cryoglobulin levels and an increase in C4 serum levels, though not to undetectable or normal levels. However, one potential concern regarding the use of such therapy is that it has a propensity to worsen HCV viraemia [141], which may lead patients to develop more severe HCVinduced liver lesions and/or cryoglobulinaemic relapses in ensuing years [147, 148]. These studies did not allow conclusions to be drawn concerning the efficacy of anti-cd20 monoclonal antibody on peripheral neuropathy and nephropathy [141, 148, 149]. The absence of efficacy on HCV viral clearance and furthermore, the potential increase in HCV viral load stresses the need for combined antiviral therapy to block the HCV infection trigger. Therapeutic guidelines In general, it appears logical that aggressive antiviral therapy with Peg-IFN and weight-based ribavirin be considered as induction Mild-to-Moderate disease (Purpura, arthralgia, polyneuropathy) Severe disease Progressive renal disease, mononeuritis multiplex, skin ulcer) Life threatening (Rapidly progressive nephritis, CNS, digestive and/or pulmonary involvement) Peg I FN-α + Ribavirin Rituximab Peg I FN-α+ Ribavirin Steroids, plasma exchange, cyclophosphamide and/or rituximab. PegIFN-α+ Ribavirin (differed) FIG. 2. Therapeutic strategies in patients with HCV-associated MC vasculitis. CNS, central nervous system.

1240 D. Saadoun et al. therapy for HCV-MC with mild-to-moderate disease severity and activity (i.e. without rapidly progressive nephritis, motor neuropathy or other life-threatening complications) (Figure 2). The duration of therapy has not yet been rigorously determined but treatment courses longer than those estimated merely based on genotype alone appear more likely to be effective [135]. The current treatment duration in HCV-MC is 12 months for all HCV genotypes with the Peg-IFN and ribavirin combination [138]. With this strategy patients with mild or moderate disease (i.e. arthralgia, purpura, sensory-motor polyneuropathy and/or isolated proteinuria) may be able to be managed without immunosuppressive agents. In patients presenting with severe disease (i.e. worsening of renal function, mononeuritis multiplex, extensive skin disease including ulcers and distal necrosis), an induction phase of immunosuppression is often necessary while awaiting the generally slow response to antiviral treatments (Figure 2). Combination therapy with rituximab and Peg-IFN- plus ribavirin appears logical and may target both the viral trigger (HCV) and the downstream B-cell arm of autoimmunity. Biologic therapy with B cell directed therapy is promising in the treatment of HCV-MC but many questions exist regarding the appropriate position of this strategy in treatment. In this setting, as with the use of rituximab in the treatment of other autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus, the duration of effect appears finite with response durations typically lasting 6 12 months and its combination with antiviral drugs is necessary. The continued efficacy and safety of repeated therapy in HCV-MC needs further investigation. For patients presenting with the most fulminant presentations including peripheral necrosis of extremities, rapidly progressive nephritis, digestive, pulmonary and/or central nervous system involvement and or signs and symptoms of hyperviscosity, apheresis can have immediate beneficial effects but must be combined with immunosuppression (cytotoxic agents, steroids) to avoid post-apheresis rebound of MC. Antiviral therapy with Peg-IFN- and ribavirin combination should be differed after the critical phase (Figure 2). References 1 Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001;345:41 52. 2 Wintrobe MM, Buell MV. Hyperproteinemia associated with multiple myeloma. Bull Johns Hopkins Hosp 1933;52:156. 3 Meltzer M, Franklin EC, Elias K, McCluskey RT, Cooper N. Cryoglobulinemia a clinical and laboratory study. II. Cryoglobulins with rheumatoid factor activity. Am J Med 1966;40:837 56. 4 Levo Y, Gorevic PD, Kassab HJ, Zucker-Franklin D, Franklin EC. Association between hepatitis B virus and essential mixed cryoglobulinemia. N Engl J Med 1977;296:1501 04. 5 Bombardieri S, Ferri C, Di Munno O, Pasero G. Liver involvement in essential mixed cryoglobulinemia. Ric Clin Lab 1979;9:361 8. 6 Popp JW, Jr, Dienstag JL, Wands JR, Bloch KJ. Essential mixed cryoglobulinemia without evidence for hepatitis B virus infection. Ann Intern Med 1980;92:379 83. 7 Galli M, Monti G, Invernizzi F et al. Hepatitis B virus-related markers in secondary and in essential mixed cryoglobulinemias: a multicentric study of 596 cases. The Italian Group for the Study of Cryoglobulinemias (GISC). Ann Ital Med Int 1992;7:209 14. 8 Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cdna clone derived from a blood-borne non-a, non-b viral hepatitis genome. Science 1989;244:359 62. 9 Pascual M, Perrin L, Giostra E, Schifferli JA. Hepatitis C virus in patients with cryoglobulinemia type II. J Infect Dis 1990;162:569 70. 10 Cacoub P, Poynard T, Ghillani P et al. Extrahepatic manifestations of chronic hepatitis C. MULTIVIRC Group. Multidepartment Virus C. Arthritis Rheum 1999;42:2204 12. 11 Nagasaka A, Takahashi T, Sasaki T et al. Cryoglobulinemia in Japanese patients with chronic hepatitis C virus infection: host genetic and virological study. J Med Virol 2001;65:52 7. 12 Donada C, Crucitti A, Donadon V et al. Systemic manifestations and liver disease in patients with chronic hepatitis C and type II or III mixed cryoglobulinaemia. J Viral Hepat 1998;5:179 85. 13 Lunel F, Musset L, Cacoub P et al. Cryoglobulinemia in chronic liver diseases: role of hepatitis C virus and liver damage. Gastroenterology 1994;106:1291 300. 14 Brouet JC, Clauvel JP, Danon F, Klein M, Seligmann M. Biologic and clinical significance of cryoglobulins. A report of 86 cases. Am J Med 1974;57:775 88. 15 Pawlotsky JM, Ben Yahia M, Andre C et al. Immunological disorders in C virus chronic active hepatitis: a prospective case-control study. Hepatology 1994;19:841 8. 16 Cacoub P, Renou C, Rosenthal E et al. Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC. Groupe d Etude et de Recherche en Medecine Interne et Maladies Infectieuses sur le Virus de l Hepatite C. Medicine (Baltimore) 2000;79:47 56. 17 Yee LJ, Kelleher P, Goldin RD et al. Antinuclear antibodies (ANA) in chronic hepatitis C virus infection: correlates of positivity and clinical relevance. J Viral Hepat 2004;11:459 64. 18 Stroffolini T, Colloredo G, Gaeta GB et al. Does an autoimmune profile affect the clinical profile of chronic hepatitis C? An Italian multicentre survey. J Viral Hepat 2004;11:257 62. 19 Monti G, Galli M, Invernizzi F et al. Cryoglobulinaemias: a multi-centre study of the early clinical and laboratory manifestations of primary and secondary disease. GISC. Italian Group for the Study of Cryoglobulinaemias. QJM 1995;88:115 26. 20 Dupin N, Chosidow O, Lunel F et al. Essential mixed cryoglobulinemia. A comparative study of dermatologic manifestations in patients infected or noninfected with hepatitis C virus. Arch Dermatol 1995;131:1124 7. 21 Leone N, Pellicano R, Ariata Maiocco I et al. Mixed cryoglobulinaemia and chronic hepatitis C virus infection: the rheumatic manifestations. J Med Virol 2002;66:200 3. 22 Lee YH, Ji JD, Yeon JE, Byun KS, Lee CH, Song GG. Cryoglobulinaemia and rheumatic manifestations in patients with hepatitis C virus infection. Ann Rheum Dis 1998;57:728 31. 23 Sene D, Ghillani-Dalbin P, Limal N et al. Anti-cyclic citrullinated peptide antibodies in hepatitis C virus associated rheumatological manifestations and Sjogren s syndrome. Ann Rheum Dis 2006;65:394 7. 24 Wener MH, Hutchinson K, Morishima C, Gretch DR. Absence of antibodies to cyclic citrullinated peptide in sera of patients with hepatitis C virus infection and cryoglobulinemia. Arthritis Rheum 2004;50:2305 8. 25 Cacoub P, Saadoun D, Limal N, Leger JM, Maisonobe T. Hepatitis C virus infection and mixed cryoglobulinaemia vasculitis: a review of neurological complications. Aids 2005;19(Suppl. 3):S128 34. 26 Authier FJ, Bassez G, Payan C et al. Detection of genomic viral RNA in nerve and muscle of patients with HCV neuropathy. Neurology 2003;60:808 12. 27 Petty GW, Duffy J, Houston J,III. Cerebral ischemia in patients with hepatitis C virus infection and mixed cryoglobulinemia. Mayo Clin Proc 1996;71:671 8. 28 Heckmann JG, Kayser C, Heuss D, Manger B, Blum HE, Neundorfer B. Neurological manifestations of chronic hepatitis C. J Neurol 1999;246:486 91. 29 Casato M, Saadoun D, Marchetti A et al. Central nervous system involvement in hepatitis C virus cryoglobulinemia vasculitis: a multicenter case-control study using magnetic resonance imaging and neuropsychological tests. J Rheumatol 2005;32:484 8. 30 Gorevic PD, Kassab HJ, Levo Y et al. Mixed cryoglobulinemia: clinical aspects and long-term follow-up of 40 patients. Am J Med 1980;69:287 308. 31 Dawson TM, Starkebaum G. Isolated central nervous system vasculitis associated with hepatitis C infection. J Rheumatol 1999;26:2273 6. 32 Mazzaro C, Tulissi P, Moretti M et al. Clinical and virological findings in mixed cryoglobulinaemia. J Intern Med 1995;238:153 60. 33 D Amico G, Fornasieri A. Cryoglobulinemic glomerulonephritis: a membranoproliferative glomerulonephritis induced by hepatitis C virus. Am J Kidney Dis 1995;25:361 9. 34 Beddhu S, Bastacky S, Johnson JP. The clinical and morphologic spectrum of renal cryoglobulinemia. Medicine (Baltimore) 2002;81:398 409. 35 D Amico G. Renal involvement in hepatitis C infection: cryoglobulinemic glomerulonephritis. Kidney Int 1998;54:650 71. 36 Mazzaro C, Pozzato G, Zorat F et al. Cryoglobulinaemic membranoproliferative glomerulonephritis and hepatitis C virus infection. Ital J Gastroenterol Hepatol 1999;31:45 53. 37 Tarantino A, Campise M, Banfi G et al. Long-term predictors of survival in essential mixed cryoglobulinemic glomerulonephritis. Kidney Int 1995;47:618 23. 38 Fornasieri A, D Amico G. Type II mixed cryoglobulinaemia, hepatitis C virus infection, and glomerulonephritis. Nephrol Dial Transplant 1996;11(Suppl. 4):25 30. 39 Levo Y, Gorevic PD, Kassab HJ, Tobias H, Franklin EC. Liver involvement in the syndrome of mixed cryoglobulinemia. Ann Intern Med 1977;87:287 92. 40 Kayali Z, Buckwold VE, Zimmerman B, Schmidt WN. Hepatitis C, cryoglobulinemia, and cirrhosis: a meta-analysis. Hepatology 2002;36:978 85. 41 Saadoun D, Asselah T, Resche-Rigon M et al. Cryoglobulinemia is associated with steatosis and fibrosis in chronic hepatitis C. Hepatology 2006;43:1337 45. 42 Moschella CM, Palmieri I, Bartolucci P, Assenza M, Maiuolo A, Modini C. Spontaneous rectus sheath haematoma in HCV mixed cryoglobulinemia requiring emergency treatment (case report). G Chir 2002;23:331 3. 43 Manganelli P, Salaffi F, Subiaco S et al. Bronchoalveolar lavage in mixed cryoglobulinaemia associated with hepatitis C virus. Br J Rheumatol 1996;35:978 82. 44 Ferri C, La Civita L, Fazzi P et al. Interstitial lung fibrosis and rheumatic disorders in patients with hepatitis C virus infection. Br J Rheumatol 1997;36:360 5. 45 Matsumori A, Ohashi N, Hasegawa K et al. Hepatitis C virus infection and heart diseases: a multicenter study in Japan. Jpn Circ J 1998;62:389 91. 46 Ramos-Casals M, Loustaud-Ratti V, De Vita S et al. Sjogren syndrome associated with hepatitis C virus: a multicenter analysis of 137 cases. Medicine (Baltimore) 2005;84:81 9. 47 Rosner I, Rozenbaum M, Toubi E, Kessel A, Naschitz JE, Zuckerman E. The case for hepatitis C arthritis. Semin Arthritis Rheum 2004;33:375 87.

HCV-related MC 1241 48 Ramos-Casals M, Cervera R, Lagrutta M et al. Clinical features related to antiphospholipid syndrome in patients with chronic viral infections (hepatitis C virus/hiv infection): description of 82 cases. Clin Infect Dis 2004;38:1009 16. 49 Haddad J, Deny P, Munz-Gotheil C et al. Lymphocytic sialadenitis of Sjogren s syndrome associated with chronic hepatitis C virus liver disease. Lancet 1992;339:321 3. 50 Garcia-Carrasco M, Font Franco J, Ingelmo Morin M, Ramos-Casals M. [Clinical manifestations and immunology associated with chronic infection with hepatitis C virus]. Rev Clin Esp 2002;202:224 32. 51 Verbaan H, Carlson J, Eriksson S et al. Extrahepatic manifestations of chronic hepatitis C infection and the interrelationship between primary Sjogren s syndrome and hepatitis C in Swedish patients. J Intern Med 1999;245:127 32. 52 El-Serag HB, Hampel H, Yeh C, Rabeneck L. Extrahepatic manifestations of hepatitis C among United States male veterans. Hepatology 2002;36:1439 45. 53 Cacoub P, Ratziu V, Myers RP et al. Impact of treatment on extra hepatic manifestations in patients with chronic hepatitis C. J Hepatol 2002;36:812 8. 54 Doffoel-Hantz V, Loustaud-Ratti V, Ramos-Casals M et al. [Evolution of Sjogren syndrome associated with hepatitis C virus when chronic hepatitis C is treated by interferon or the association of interferon and ribavirin]. Rev Med Interne 2005;26:88 94. 55 Ramos-Casals M, Garcia-Carrasco M, Cervera R et al. Hepatitis C virus infection mimicking primary Sjogren syndrome. A clinical and immunologic description of 35 cases. Medicine (Baltimore) 2001;80:1 8. 56 Koike K, Moriya K, Ishibashi K et al. Sialadenitis histologically resembling Sjogren syndrome in mice transgenic for hepatitis C virus envelope genes. Proc Natl Acad Sci USA 1997;94:233 6. 57 Sene D, Ghillani-Dalbin P, Thibault V et al. Longterm course of mixed cryoglobulinemia in patients infected with hepatitis C virus. J Rheumatol 2004;31:2199 206. 58 Saadoun D, Bieche I, Maisonobe T et al. Involvement of chemokines and type 1 cytokines in the pathogenesis of hepatitis C virus-associated mixed cryoglobulinemia vasculitis neuropathy. Arthritis Rheum 2005;52:2917 25. 59 Boyer O, Saadoun D, Abriol J et al. CD4þCD25þ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis. Blood 2004;103:3428 30. 60 Cacoub P, Renou C, Kerr G et al. Influence of HLA-DR phenotype on the risk of hepatitis C virus-associated mixed cryoglobulinemia. Arthritis Rheum 2001;44:2118 24. 61 Bianchettin G, Bonaccini C, Oliva R et al. Analysis of hepatitis C virus hypervariable region 1 sequence in cryoglobulinaemic patients and associated controls. J Virol 2007;81:4564 71. 62 Vallat L, Benhamou Y, Gutierrez M et al. Clonal B cell populations in the blood and liver of patients with chronic hepatitis C virus infection. Arthritis Rheum 2004;50:3668 78. 63 Ferri C, Sebastiani M, Giuggioli D et al. Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients. Semin Arthritis Rheum 2004;33:355 74. 64 Ramos-Casals M, Robles A, Brito-Zeron P et al. Life-threatening cryoglobulinemia: clinical and immunological characterization of 29 cases. Semin Arthritis Rheum 2006;36:189 96. 65 Rasul I, Shepherd FA, Kamel-Reid S, Krajden M, Pantalony D, Heathcote EJ. Detection of occult low-grade b-cell non-hodgkin s lymphoma in patients with chronic hepatitis C infection and mixed cryoglobulinemia. Hepatology 1999;29: 543 7. 66 Luppi M, Longo G, Ferrari MG et al. Clinico-pathological characterization of hepatitis C virus-related B-cell non-hodgkin s lymphomas without symptomatic cryoglobulinemia. Ann Oncol 1998;9:495 8. 67 Gorevic PD, Frangione B. Mixed cryoglobulinemia cross-reactive idiotypes: implications for the relationship of MC to rheumatic and lymphoproliferative diseases. Semin Hematol 1991;28:79 94. 68 Invernizzi F, Galli M, Serino G et al. Secondary and essential cryoglobulinemias. Frequency, nosological classification, and long-term follow-up. Acta Haematol 1983;70:73 82. 69 Monti G, Pioltelli P, Saccardo F et al. Incidence and characteristics of non-hodgkin lymphomas in a multicenter case file of patients with hepatitis C virus-related symptomatic mixed cryoglobulinemias. Arch Intern Med 2005;165:101 5. 70 Zuckerman E, Zuckerman T, Levine AM et al. Hepatitis C virus infection in patients with B-cell non-hodgkin lymphoma. Ann Intern Med 1997;127:423 8. 71 Germanidis G, Haioun C, Pourquier J et al. Hepatitis C virus infection in patients with overt B-cell non-hodgkin s lymphoma in a French center. Blood 1999;93:1778 9. 72 Hausfater P, Cacoub P, Rosenthal E et al. Hepatitis C virus infection and lymphoproliferative diseases in France: a national study. The GERMIVIC Group. Am J Hematol 2000;64:107 11. 73 Ferri C, Caracciolo F, Zignego AL et al. Hepatitis C virus infection in patients with non-hodgkin s lymphoma. Br J Haematol 1994;88:392 4. 74 Silvestri F, Pipan C, Barillari G et al. Prevalence of hepatitis C virus infection in patients with lymphoproliferative disorders. Blood 1996;87:4296 301. 75 Mazzaro C, Zagonel V, Monfardini S et al. Hepatitis C virus and non-hodgkin s lymphomas. Br J Haematol 1996;94:544 50. 76 Izumi T, Sasaki R, Miura Y, Okamoto H. B cell malignancy and hepatitis C virus infection. Leuk Res 1996;20:445. 77 De Rosa G, Gobbo ML, De Renzo A et al. High prevalence of hepatitis C virus infection in patients with B-cell lymphoproliferative disorders in Italy. Am J Hematol 1997;55:77 82. 78 Duberg AS, Nordstrom M, Torner A et al. Non-Hodgkin s lymphoma and other nonhepatic malignancies in Swedish patients with hepatitis C virus infection. Hepatology 2005;41:652 9. 79 Brind AM, Watson JP, Burt A et al. Non-Hodgkin s lymphoma and hepatitis C virus infection. Leuk Lymphoma 1996;21:127 30. 80 Collier JD, Zanke B, Moore M et al. No association between hepatitis C and B-cell lymphoma. Hepatology 1999;29:1259 61. 81 Hausfater P, Cacoub P, Sterkers Y et al. Hepatitis C virus infection and lymphoproliferative diseases: prospective study on 1,576 patients in France. Am J Hematol 2001;67:168 71. 82 Zuckerman E, Zuckerman T. Hepatitis C and B-cell lymphoma: the hematohepatologist linkage. Blood Rev 2002;16:119 25. 83 Gisbert JP, Garcia-Buey L, Pajares JM, Moreno-Otero R. Prevalence of hepatitis C virus infection in B-cell non-hodgkin s lymphoma: systematic review and meta-analysis. Gastroenterology 2003;125:1723 32. 84 Matsuo K, Kusano A, Sugumar A, Nakamura S, Tajima K, Mueller NE. Effect of hepatitis C virus infection on the risk of non-hodgkin s lymphoma: a meta-analysis of epidemiological studies. Cancer Sci 2004;95:745 52. 85 Vallisa D, Berte R, Rocca A et al. Association between hepatitis C virus and non-hodgkin s lymphoma, and effects of viral infection on histologic subtype and clinical course. Am J Med 1999;106:556 60. 86 Agnello V, De Rosa FG. Extrahepatic disease manifestations of HCV infection: some current issues. J Hepatol 2004;40:341 52. 87 Mele A, Pulsoni A, Bianco E et al. Hepatitis C virus and B-cell non-hodgkin lymphomas: an Italian multicenter case-control study. Blood 2003;102:996 9. 88 Trejo O, Ramos-Casals M, Lopez-Guillermo A et al. Hematologic malignancies in patients with cryoglobulinemia: association with autoimmune and chronic viral diseases. Semin Arthritis Rheum 2003;33:19 28. 89 Sakamuro D, Furukawa T, Takegami T. Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells. J Virol 1995;69:3893 6. 90 Moriya K, Fujie H, Shintani Y et al. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 1998;4:1065 7. 91 Pileri P, Uematsu Y, Campagnoli S et al. Binding of hepatitis C virus to CD81. Science 1998;282:938 41. 92 Agnello V, Abel G, Elfahal M, Knight GB, Zhang QX. Hepatitis C virus and other flaviviridae viruses enter cells via low density lipoprotein receptor. Proc Natl Acad Sci USA 1999;96:12766 71. 93 Levine AM, Shimodaira S, Lai MM. Treatment of HCV-related mantle-cell lymphoma with ribavirin and pegylated interferon Alfa. N Engl J Med 2003;349:2078 9. 94 Pal S, Sullivan DG, Kim S et al. Productive replication of hepatitis C virus in perihepatic lymph nodes in vivo: implications of HCV lymphotropism. Gastroenterology 2006;130:1107 16. 95 Ferri C, La Civita L, Zignego AL, Pasero G. Viruses and cancers: possible role of hepatitis C virus. Eur J Clin Invest 1997;27:711 8. 96 Stolte M, Bayerdorffer E, Morgner A et al. Helicobacter and gastric MALT lymphoma. Gut 2002;50(Suppl. 3):19 24. 97 Quinn ER, Chan CH, Hadlock KG, Foung SK, Flint M, Levy S. The B-cell receptor of a hepatitis C virus (HCV)-associated non-hodgkin lymphoma binds the viral E2 envelope protein, implicating HCV in lymphomagenesis. Blood 2001;98:3745 9. 98 De Re V, Sansonno D, Simula MP et al. HCV-NS3 and IgG-Fc crossreactive IgM in patients with type II mixed cryoglobulinemia and B-cell clonal proliferations. Leukemia 2006;20:1145 54. 99 Sansonno D, Lauletta G, De Re V et al. Intrahepatic B cell clonal expansions and extrahepatic manifestations of chronic HCV infection. Eur J Immunol 2004;34:126 36. 100 De Re V, De Vita S, Marzotto A et al. Sequence analysis of the immunoglobulin antigen receptor of hepatitis C virus-associated non-hodgkin lymphomas suggests that the malignant cells are derived from the rheumatoid factor-producing cells that occur mainly in type II cryoglobulinemia. Blood 2000;96:3578 84. 101 Marasca R, Vaccari P, Luppi M et al. Immunoglobulin gene mutations and frequent use of VH1 69 and VH4 34 segments in hepatitis C virus-positive and hepatitis C virus-negative nodal marginal zone B-cell lymphoma. Am J Pathol 2001;159:253 61. 102 Libra M, Capello D, Gloghini A et al. Analysis of aberrant somatic hypermutation (SHM) in non-hodgkin s lymphomas of patients with chronic HCV infection. J Pathol 2005;206:87 91. 103 Ivanovski M, Silvestri F, Pozzato G et al. Somatic hypermutation, clonal diversity, and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 1998;91:2433 42. 104 De Re V, De Vita S, Marzotto A et al. Pre-malignant and malignant lymphoproliferations in an HCV-infected type II mixed cryoglobulinemic patient are sequential phases of an antigen-driven pathological process. Int J Cancer 2000;87:211 6. 105 De Vita S, Zagonel V, Russo A et al. Hepatitis C virus, non-hodgkin s lymphomas and hepatocellular carcinoma. Br J Cancer 1998;77:2032 5. 106 Chan CH, Hadlock KG, Foung SK, Levy S. V(H)1 69 gene is preferentially used by hepatitis C virus-associated B cell lymphomas and by normal B cells responding to the E2 viral antigen. Blood 2001;97:1023 6. 107 Machida K, Cheng KT, Sung VM et al. Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes. Proc Natl Acad Sci USA 2004;101:4262 7. 108 Kitay-Cohen Y, Amiel A, Hilzenrat N et al. Bcl-2 rearrangement in patients with chronic hepatitis C associated with essential mixed cryoglobulinemia type II. Blood 2000;96:2910 2.