Patrice Cacoub
email: patrice.cacoub@aphp.fr

Cloé Comarmond

Matheus Vieira

Paul Régnier

David Saadoun

Smith Kline

Vifor David Saadoun

Cloé

Prof Patrice Cacoub

HCV-related lymphoproliferative disorders in the direct-acting antivirals era: from mixed cryoglobulinemia to B-cell lymphoma

Keywords: HCV (Hepatitis C), extrahepatic manifestation, cryoglobulinemia, vasculitis, lymphoma, direct acting-antivirals (DAA), treatment HCV, direct-acting antiviral agents, lymphoproliferative disorder, cryoglobulinemia, lymphoma

Hepatitis C virus (HCV) has been shown to induce many B-cell lymphoproliferative disorders. B lymphocytes are specialized in producing immunoglobulins, and within chronic HCV infection they can cause a range that goes from polyclonal hypergammaglobulinemia without clinical repercussions, passing by mixed cryoglobulinemic vasculitis and eventually reaching B-cell non-Hodgkin lymphoma. This spectrum is supported by substantial epidemiological, pathophysiological and therapeutic data. Many, although not all, pathogenic pathways leading from one extreme to another have been decrypted. Chronic viral antigen stimulation of B lymphocytes has a central role until the last steps towards overt malignancy. This has direct implications in treatment strategies, which always include the use of direct-acting antiviral agents sometimes associated with immunosuppressants. The role of direct-acting antiviral agents has been well established in patients with cryoglobulinemia vasculitis. Its positive impact on B cell non-Hodgkin lymphoma needs to be confirmed in larger studies with longer follow-up.

Introduction

The hepatitis C virus (HCV) is estimated to infect more than 70 million people worldwide. Although hepatotropic in essence, chronic HCV infection has been associated with many extrahepatic manifestations, which have been observed in up to two-thirds of patients [START_REF] Cacoub | Extrahepatic manifestations of chronic hepatitis C[END_REF] Circulating cryoglobulins is the most frequent extrahepatic manifestation and is present in half of patients. These cryoglobulins are the product of chronically stimulated B lymphocytes and can be seen as hypergammaglobulinemia in serum protein electrophoresis. Lymphoproliferative disorders, either benign such as cryoglobulinemia vasculitis, or malignant such as B-cell non-Hodgkin lymphoma (B-NHL), represent well-described extrahepatic manifestations of HCV and appear to represent both ends of the spectrum of systemic viral-induced disease [START_REF] Cacoub | Extrahepatic Manifestations of Chronic HCV Infection[END_REF]. Lymphocytes share the same CD81 transmembrane receptor as hepatocytes, to which the envelope viral protein E2 binds and leads to several changes in lymphocyte homeostasis. A complex multistep process supports the progression from asymptomatic cryoglobulinemia to the vasculitis clinical syndrome and ultimately to overt B-cell malignancy (3). The fact that cryoglobulinemia is the main risk factor for the development of B-NHL reinforces the link between these entities, suggesting the same process at different time points [START_REF] Monti | Incidence and Characteristics of Non-Hodgkin Lymphomas in a Multicenter Case File of Patients With Hepatitis C Virus-Related Symptomatic Mixed Cryoglobulinemias[END_REF]. Several other mechanisms and arguments have since endorsed this. The sustained virological response (SVR) following interferon alpha (IFN)-based therapeutic regimens has been proven to reduce the risk of developing both cryoglobulinemic vasculitis and B-NHL in HCV-infected patients [START_REF] Mahale | The effect of sustained virological response on the risk of extrahepatic manifestations of hepatitis C virus infection[END_REF]. With the recent advent of direct-acting antivirals (DAA), which are well-tolerated therapies with higher rates of SVR, great enthusiasm has been generated around their potential beneficial effects on extrahepatic manifestations. The purpose of this review is to describe the updated basis of the existing lymphoproliferation continuumfrom cryoglobulinemia to B-NHLwithin chronic HCV infection, with a focus on the impact of DAA on these conditions. The reader is referred to other recent and comprehensive reviews covering aspects of HCV extra-hepatic manifestations with distinct perspectives [START_REF] Cacoub | Extrahepatic Manifestations of Chronic HCV Infection[END_REF]3,[START_REF] Couronné | From hepatitis C virus infection to B-cell lymphoma[END_REF].

Epidemiology and clinical manifestations 2.1 HCV-related mixed cryoglobulinemia vasculitis

Epidemiology

Mixed cryoglobulinemia vasculitis (CryoVas) is a small-vessel systemic vasculitis.

Before the era of direct-acting antivirals, HCV was the main cause of mixed cryoglobulinemia, accounting for between 70% and 90% of cases, according to the geographical region. Available since 2014, DAA regimens provided a highly effective antiviral treatment for HCV, which modified this picture. Currently, systemic autoimmune disorders (mainly systemic lupus erythematosus and Sjögren's syndrome) and hematological malignancies appear to be the main causes of mixed cryoglobulinemia in developed countries, with HCV infection ranking third, as noted in a recent study we conducted in France [START_REF] Boleto | Cryoglobulinemia after the era of chronic hepatitis C infection[END_REF].

Main clinical features

Mixed cryoglobulinemia vasculitis may lead to clinical manifestations ranging from purpura, arthralgia and fatigue to more serious and life-threatening complications with neurologic and renal involvement (Figure 1) [START_REF] Cacoub | Extrahepatic manifestations associated with hepatitis C virus infection. A prospective multicenter study of 321 patients. The GERMIVIC[END_REF]. The main cutaneous manifestation is palpable purpura. Raynaud's phenomenon, acrocyanosis, skin ulcers and chronic ulcerations of the lower limbs can also occur. Joint involvement commonly appears as non-erosive arthralgia that affects large joints, whereas arthritis is rare. The most frequent neurological manifestation is distal sensory or sensory-motor polyneuropathy.

Multiple mononeuropathy may occur less frequently. Regarding renal involvement,

HCV-related lymphomas

Epidemiology

An association between HCV infection and B-NHL has been reported since the virus was first identified, and observations from several studies have been confirmed in metaanalyses. Gisbert et al. (9) pooled data from 5542 patients and estimated the HCV infection prevalence to be 15% among B-NHL patients, which is considerably higher than that observed in the general population (1.5%) and in patients with other hematologic malignancies (2.9%). Another meta-analysis comprising 15 case-control and 3 prospective studies yielded a relative risk (RR) for B-cell NHL among HCV patients of 2.5 (95% CI 2.1-3.0). A striking geographic variation in B-NHL prevalence was noted, as the RR in countries with high HCV prevalence ( 5%) was 3.01, while in those with low prevalence it was 1.9. Overall, it has been estimated that HCV could account for up to 10% of B-NHL cases in countries with high HCV prevalence, whereas it is less than 1% in low-prevalence countries.

Another argument for the role of HCV in lymphomagenesis came from early reports and case series that associated SVR achievement to lymphoma regression, as well as the recurrence of positive viremia to lymphoma relapse [START_REF] Casato | Regression of lymphoproliferative disorder after treatment for hepatitis C virus infection in a patient with partial trisomy 3, Bcl-2 overexpression, and type II cryoglobulinemia[END_REF][START_REF] Saadoun | Splenic lymphoma with villous lymphocytes, associated with type II cryoglobulinemia and HCV infection: a new entity?[END_REF]. If HCV is considered at the origin of B-NHL, viral eradication should have a favorable impact on lymphoma development over time. Early positive results derived from a large cohort of HCV patients treated with IFN-based therapies demonstrated a reduction in B-NHL incidence in those who reached SVR (HR = 0.13) [START_REF] Kawamura | Viral elimination reduces incidence of malignant lymphoma in patients with hepatitis C[END_REF]. The annual incidence of B-NHL among HCV patients was estimated as 0.23%. The main limitation of such results is related to the well-known immunomodulation properties of IFN, which may have induced B-NHL regression regardless of its antiviral effect. This trend has been further confirmed in recent large retrospective studies. A nationwide Taiwanese real-world cohort evaluated 10,714 patients who had received IFN-based therapy and were followed over a median of 3.79 years [START_REF] Huang | Extrahepatic Malignancy Among Patients With Chronic Hepatitis C After Antiviral Therapy: A Real-World Nationwide Study on Taiwanese Chronic Hepatitis C Cohort (T-COACH)[END_REF]. Patients not reaching an SVR had a significantly higher risk of developing B-NHL than those with an SVR (0.08% vs. 0.03% per person-year, respectively; p = 0.03). Notably, this effect was seen only in patients younger than 65 years, reinforcing the prospect that early anti-viral treatment should be the rule. In an even larger cohort with 69,581 HCV American veterans (predominantly males), an IFNinduced SVR was able to significantly reduce the B-NHL incidence over a mean follow-up period of 10.6 years [adjusted hazard ratio (aHR) = 0.70; 95% CI 0.51-0.97] [START_REF] Dal Maso | Hepatitis C virus and risk of lymphoma and other lymphoid neoplasms: a meta-analysis of epidemiologic studies[END_REF]. Interestingly, a reduction in the combined incidence of all B-cell hematologic malignancies (including multiple myeloma) and monoclonal gammopathy of undetermined significance was also noted (aHR, 0.67; 95% CI 0.53-0.84). Although a DAA-induced SVR was not shown to have the same benefit in this study, it is still not possible to draw definite conclusions due to a shorter follow-up as compared with IFNtreated patients (2.9 vs. 10.6 years, respectively).

B cell lymphoma subtypes

Different types of B-NHL have been associated with HCV infection. An early metaanalysis found a consistently higher risk for all B-NHL subtypes [START_REF] Dal Maso | Hepatitis C virus and risk of lymphoma and other lymphoid neoplasms: a meta-analysis of epidemiologic studies[END_REF]. However, the strongest associations were with low-grade B-NHLs occurring in the spleen or liver, namely marginal zone lymphoma (MZL) and lymphoplasmacytic lymphoma (LL), and with high-grade diffuse large B-cell lymphoma (DLBCL). A large international casecontrol study found an odds ratio (OR) of 2.47 (95% CI 1.44-4.23), 2.57 (95% CI 1.14-5.79) and 2.24 (95% CI 1.68-2.99) for these three subtypes, respectively [START_REF] Sanjose S De | Hepatitis C and non-Hodgkin lymphoma among 4784 cases and 6269 controls from the International Lymphoma Epidemiology Consortium[END_REF]. Other low-grade B-NHLs, such as follicular lymphoma (FL) and chronic lymphocytic leukemia, were not associated with HCV. A large American case-control study confirmed the association for MZL and DLBCL [START_REF] Anderson | Hematopoietic malignancies associated with viral and alcoholic hepatitis[END_REF]. HCV-associated DLBCL can occur either as de novo or from MZL transformation. A multicentric study demonstrated that DLBCL in HCV-positive patients most frequently arose from transformed MZL when compared with those without HCV (32% vs. 6%, p = 0.02), endorsing the continuum that exists in HCV-induced lymphoproliferation [START_REF] Paulli | Subcutaneous "lipoma-like" B-cell lymphoma associated with HCV infection: a new presentation of primary extranodal marginal zone B-cell lymphoma of MALT[END_REF]. Some peculiar B-NHL subtypes have been specifically associated with HCV in case series, such as splenic MZL with villous lymphocytes [START_REF] Saadoun | Splenic lymphoma with villous lymphocytes, associated with type II cryoglobulinemia and HCV infection: a new entity?[END_REF] and subcutaneous 'lipomalike' extranodal MZL [START_REF] Paulli | Subcutaneous "lipoma-like" B-cell lymphoma associated with HCV infection: a new presentation of primary extranodal marginal zone B-cell lymphoma of MALT[END_REF], which is apparently responsive to antivirals. A monoclonal gammopathy, mainly IgMk, has also been reported in HCV-infected patients [START_REF] Andreone | Prevalence of monoclonal gammopathies in patients with hepatitis C virus infection[END_REF]. A large US Veterans Affairs study found that HCV promoted a 3-fold higher risk for Waldenström macroglobulinemia, which was not found for multiple myeloma [START_REF] Giordano | Risk of non-Hodgkin lymphoma and lymphoproliferative precursor diseases in US veterans with hepatitis C virus[END_REF].

Clinical presentations

There are several features that make the lymphoma presentation peculiar among HCV patients. First, the age of onset seems lower in HCV-infected patients, as B-cell NHL was reported to be diagnosed on average 4.6 years earlier than those without the virus [START_REF] Allison | Increased incidence of cancer and cancer-related mortality among persons with chronic hepatitis C infection, 2006-2010[END_REF]. Among HCV patients, the presence of CryoVas is by far the strongest risk factor for developing B-NHL, with a 35 times greater risk than that of the general population [START_REF] Monti | Incidence and Characteristics of Non-Hodgkin Lymphomas in a Multicenter Case File of Patients With Hepatitis C Virus-Related Symptomatic Mixed Cryoglobulinemias[END_REF]. Other reported risk factors include female gender, long lasting infection and being from an HCV endemic region [START_REF] Ferri | Expert opinion on managing chronic HCV in patients with non-Hodgkin lymphoma and other extrahepatic malignancies[END_REF]. As for the clinical presentation, splenomegaly or extranodal involvement (i.e. liver, salivary glands) have been reported more often in HCV-positive than in HCV-negative patients [START_REF] Vita | Characterization of overt B-cell lymphomas in patients with hepatitis C virus infection[END_REF]. Infected patients were also shown to have more frequent monoclonal gammopathy and cryoglobulinemia (probable consequences of long-lasting B cell activation). All these characteristics were recently reproduced in a large prospective multicenter cohort, and highlighted that autoimmune phenomena are more common among indolent than aggressive lymphomas [START_REF] Rattotti | Lymphomas associated with chronic hepatitis C virus infection: A prospective multicenter cohort study from the Rete Ematologica Lombarda (REL) clinical network[END_REF].

This distinct clinical presentation also reflects a worse prognosis and challenging treatment. A large American cohort revealed higher mortality rates for HCV-infected patients with B-NHL, while death occurred on average 12.6 years earlier than for those without HCV infection [START_REF] Allison | Increased incidence of cancer and cancer-related mortality among persons with chronic hepatitis C infection, 2006-2010[END_REF]. Regarding transformed DLBCL, HCV-infected patients had more relapses and a worse 2-year survival rate than those who were not infected [START_REF] Hosry | Clinicopathologic characteristics and outcomes of transformed diffuse large B-cell lymphoma in hepatitis C virus-infected patients[END_REF]. This study also reported that CD5 + B cellsa known marker of poor prognosis were more frequently seen in HCV-infected than non-infected patients, and their presence was closely associated with B-NHL relapse after chemotherapy. Regarding other treatment modalities, HCV-NHL patients undergoing hematopoietic cell transplantation were reported to progress faster to cirrhosis than those not transplanted [START_REF] De Latour | Long-term outcome of hepatitis C infection after bone marrow transplantation[END_REF].

Pathophysiology

Benign lymphoproliferation: cryoglobulinemic vasculitis

Cryoglobulinemia is defined as the presence in the serum of an immunoglobulin (Ig) that precipitates in vitro at 4°C and dissolves at 37°C. Circulating mixed cryoglobulins are detected in 40% to 60% of patients who are chronically infected with HCV, while HCV-CryoVas occurs in approximately 15% of HCV-infected patients. Mixed cryoglobulinemia is characterized by the presence of type II and type III cryoglobulins, which consist of polyclonal IgG with monoclonal and polyclonal IgM, respectively, both with rheumatoid factor activity (RF). During follow-up, biological improvement is assessed by the serum level of cryoglobulinemia and surrogate markers (C4 serum level and RF).

The mechanisms that lead to HCV-induced cryoglobulinemia and the reasons that cryoglobulinemia can be symptomatic or asymptomatic are not fully understood.

Numerous factors support the autoimmune origin of HCV-related CryoVas, such as the presence of autoantibodies (mixed cryoglobulin with RF activity), the presence of T-cell infiltrates in small-sized vessels, and the observation that specific HLA alleles confer susceptibility to CryoVas (3,[START_REF] Boleto | Hepatitis C virus-related vasculitis[END_REF]. The pathophysiological cellular responses guiding this autoimmunity are summarized in Figure 3. HCV interacts directly with lymphocytes and leads to modulation of B-and T-cell function, which then leads to polyclonal activation and expansion of B cells producing RF [START_REF] Cacoub | Extrahepatic manifestations of chronic hepatitis C[END_REF][START_REF] Saadoun | Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation[END_REF]. Abnormal T cell homeostasis is observed in HCV-CryoVas patients, with a quantitative defect in regulatory T cells (Tregs), and Th1 polarization [START_REF] Boyer | CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis[END_REF][START_REF] Saadoun | Predominance of type 1 (Th1) cytokine production in the liver of patients with HCVassociated mixed cryoglobulinemia vasculitis[END_REF]. The benign B-cell lymphoproliferation likely results from an indirect process following the chronic antigenic stimulation of a limited pool of preexisting autoreactive B cells. It has been proposed that persistently high levels of HCV-containing immune complexes stimulate the proliferation of RF-bearing B cells, but the precise antigen and stimulatory mechanisms have remained elusive.

Clonal expansion of CD27 + IgM + CD21 low/-memory B cells, referred to as activated or atypical memory B cells (AtMs), has been described in HCV-CryoVas patients [START_REF] Terrier | Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infection-related autoimmunity[END_REF][START_REF] Charles | Clonal expansion of immunoglobulin M+CD27+ B cells in HCV-associated mixed cryoglobulinemia[END_REF][START_REF] Comarmond | TLR9 signalling in HCV-associated atypical memory B cells triggers Th1 and rheumatoid factor autoantibody responses[END_REF].

AtMs are clonal or clonally-related, and mainly express the VH1-69 IgH gene, which is also highly prevalent in HCV-associated malignant lymphoproliferations. Low expression of CD21 on these clonal memory B-cells reflects an anergic state [START_REF] Isnardi | Complement receptor 2/CD21-human naive B cells contain mostly autoreactive unresponsive clones[END_REF]. AtMs are prone to undergo apoptosis and do not proliferate upon BCR stimulation, but they respond to the TLR9 agonist CpG by expressing activator and proliferative markers.

Anergy is a well-known regulatory mechanism for maintaining immune tolerance of autoreactive cells. AtMs in HCV-CryoVas produce somatically mutated RF autoantibodies and are not removed from the B cell repertoire. It remains unclear why anergy mechanisms fail to prevent NHL development in some HCV-CryoVas patients.

Interestingly, in HCV-CryoVas patients treated with DAAs, the numbers of regulatory T cells increased, IgM + CD21 -/low memory B cells decreased, and T follicular helper (Tfh) cells decreased, restoring disturbances in peripheral B-and T-cell homeostasis [START_REF] Comarmond | Direct-Acting Antiviral Therapy Restores Immune Tolerance to Patients With Hepatitis C Virus-Induced Cryoglobulinemia Vasculitis[END_REF]. DAA therapy also appears to exert an effect on B-cell phenotype by reducing the proportion of circulating CD21 -/low AtMs. Despite this positive impact, B clones may persist in the serum after DAA-induced SVR, but so far, their persistence has not been correlated with reactivation of vasculitis [START_REF] Visentini | Long-lasting persistence of large B-cell clones in hepatitis C virus-cured patients with complete response of mixed cryoglobulinaemia vasculitis[END_REF]. While the mechanisms behind CryoVas relapses after DAA remain unclear, biomarkers such as monoclonal B-cell lymphocytosis, t(14;18) translocation, and abnormal free light chains κ/λ ratios have been proposed as biological elements to foresee clinical response [START_REF] Gragnani | Hematological and Genetic Markers in the Rational Approach to Patients With HCV Sustained Virological Response With or Without Persisting Cryoglobulinemic Vasculitis[END_REF].

Malignant lymphoproliferation: lymphoma

Despite not coding for oncogenes and being unable to be integrated into the host genome [START_REF] Machida | Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes[END_REF], HCV appears to be involved in lymphomagenesis through diverse nonexclusive mechanisms. As for the lymphotropism seen in CryoVas, the continuous exposure of B-cell receptors to viral antigens plays a major role in malignant clonal expansion, mainly in the MZL subtype, but also in transformed DLBCL [START_REF] Vannata | Hepatitis C virus-associated B-cell non-Hodgkin's lymphomas: what do we know?[END_REF]. CD81 engagement with E2 viral protein has been shown to reduce the B-cell threshold for activation and proliferation, either in isolation [START_REF] Rosa | Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virusassociated B lymphocyte disorders[END_REF] or in combination with BCR binding along with the CD19/CD21/CD81 complex [START_REF] Weng | Hepatitis C virus (HCV) and lymphomagenesis[END_REF]. These interactions are able to directly induce immunoglobulin gene hypermutations, notably in the VH region as described above [START_REF] Rosa | Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virusassociated B lymphocyte disorders[END_REF]. The high mutation rate may lead to the production of HCV antibodies with lower affinity and specificity, which favor viral escape from immune surveillance [START_REF] Machida | Hepatitis C virus (HCV)-induced immunoglobulin hypermutation reduces the affinity and neutralizing activities of antibodies against HCV envelope protein[END_REF].

E2-CD81 binding could also lead to bcl-2 rearrangement and t(14;18) translocationknown apoptosis inhibitorsas shown in HCV patients with MALT lymphomas and CryoVas [START_REF] Libra | Association of t(14;18) translocation with HCV infection in gastrointestinal MALT lymphomas[END_REF][START_REF] Zignego | Prevalence of bcl-2 rearrangement in patients with hepatitis C virus-related mixed cryoglobulinemia with or without B-cell lymphomas[END_REF]. Besides CD81, the co-stimulatory receptor B7.2 (CD86) mediates lymphotropism through inhibition of memory B-cell function, and thus enhances persistent infection in hosts [START_REF] Chen | Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2[END_REF]. HCV core protein is another implicated viral protein, as it could induce B-cell proliferation through interleukin-6 (IL-6) production by CD14 + cells from HCV-NHL patients [START_REF] Feldmann | Induction of interleukin-6 by hepatitis C virus core protein in hepatitis C-associated mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma[END_REF]. B-lymphocyte stimulator (BLyS), another strong Bcell stimulating cytokine, has also been associated with HCV-NHL [START_REF] Sène | Hepatitis C virus-associated B-cell proliferation--the role of serum B lymphocyte stimulator (BLyS/BAFF)[END_REF]. These data support the stepwise process involved in HCV-induced lymphoproliferation and suggest additional required mechanisms in the transformation to overt lymphoma.

Although HCV is capable of infecting lymphocytes, its ability to replicate within these cells in vivo does not appear as vigorous as within hepatocytes [START_REF] Machida | Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes[END_REF]. The so-called "hit and run" theory has been proposed to explain further steps involved in B-cell damage beyond persistent intracellular replication. A mutator phenotype is induced by HCV in vitro, increasing mutation frequency in proto-oncogenes and tumor suppressor genes, such as bcl-6, p53 and beta-catenin. This is further enhanced by the induced doublestrand breaks through activation of error-prone polymerases and activation-induced cytidine deaminase (AID) [START_REF] Machida | Hepatitis C virus induces a mutator phenotype: enhanced mutations of immunoglobulin and protooncogenes[END_REF]. Another involved mechanism is related to nitric oxide (NO) production, with oxidative stress causing mitochondrial and DNA damage [START_REF] Machida | Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to DNA damage and STAT3 activation[END_REF].

Core protein and non-structural protein 3 (NS3) expression have also been associated with oxidative stress induction [START_REF] Machida | Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes[END_REF].

The roles of viral particles and intracellular networks have been increasingly emphasized. NS4B protein has been shown to play a critical part in the activation of bcl-2 and matrix metalloproteinase-2 through multiple cellular signaling pathways, including several members of the protein kinase C (PKC) family: JNK, ERK and STAT3 [START_REF] Li | Hepatitis C virus activates Bcl-2 and MMP-2 expression through multiple cellular signaling pathways[END_REF]. Of note, the JNK pathway was also reported be induced through E2-CD81 engagement [START_REF] Rosa | Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virusassociated B lymphocyte disorders[END_REF]. NS3/4A protein overexpression ex vivo was recently demonstrated to have a role in posttranscriptional regulation of target mRNAs, increasing BCR signaling from inside B cell [START_REF] Dai | Hepatitis C virus upregulates B-cell receptor signaling: a novel mechanism for HCV-associated Bcell lymphoproliferative disorders[END_REF]. Besides viral particles, the role of microRNAs (miRNAs) has been highlighted in the intracellular networks' regulation. Downregulation of some miRNA were demonstrated to drive lymphomagenesis in HCV-transgenic mice and activate both canonical and alternative NF-κB pathways [START_REF] Kasama | B-cell-intrinsic hepatitis C virus expression leads to B-cell-lymphomagenesis and induction of NF-κB signalling[END_REF]. The role of miRNAs networks is further reinforced by distinct signatures reported in splenic tissue from MZL patients (53); they have also been found to be potential biomarkers in lymph node samples from DLBCL [START_REF] Augello | MicroRNA as potential biomarker in HCV-associated diffuse large B-cell lymphoma[END_REF]. In turn, the transition between these low and high-grade subtypes remains poorly understood but has been linked to dysregulation in NOTCH signaling. This pathway, which is known to be involved in MZL pathogenesis, was shown to have a significantly higher mutation frequency in HCV-positive transformed DLBCL patients than in their negative counterparts [START_REF] Arcaini | The NOTCH pathway is recurrently mutated in diffuse large B-cell lymphoma associated with hepatitis C virus infection[END_REF].

There are several mechanisms involved in the HCV lymphoproliferation continuum, regardless of the viral presence within B cells. The processes leading to overt lymphoma are shown in Figure 4. These are not necessarily mutually exclusive but instead seem to flow in parallel, increasing the oncogenic potential. Of note, if HCV is indeed able to cause permanent B cell damage, a no-return point in lymphoproliferative pathogenesis could limit antiviral efficacy.

Therapeutic strategies 4.1 HCV-related cryoglobulinemic vasculitis

DAA have drastically changed the treatment of chronic HCV infection [START_REF] Liver | Electronic address: easloffice@easloffice.eu, Clinical Practice Guidelines Panel: Chair:, EASL Governing Board representative:, Panel members: EASL recommendations on treatment of hepatitis C: Final update of the series☆[END_REF]. DAA induce SVR rates greater than 95% with very rare and minor side effects. They have also proven efficacious for extrahepatic manifestations such as CryoVas. In the largest prospective study, including 148 patients with HCV-CryoVas, a complete clinical response of vasculitis manifestations was observed in 73% of patients (106/148), a partial response in 23% (33/148) and no response in 5% (7/148) [START_REF] Cacoub | Long-term Efficacy of Interferon-Free Antiviral Treatment Regimens in Patients With Hepatitis C Virus-Associated Cryoglobulinemia Vasculitis[END_REF]. Cryoglobulinemia resolved in more than half of patients [START_REF] Cacoub | Long-term Efficacy of Interferon-Free Antiviral Treatment Regimens in Patients With Hepatitis C Virus-Associated Cryoglobulinemia Vasculitis[END_REF]. A complete response was observed for purpura in 97% of patients, renal involvement in 91%, arthralgias in 86% and neuropathy in 77%. The severity of CryoVas and peripheral neuropathy were associated with no or partial response. A therapeutic strategy for HCV-associated renal disease includes DAA combinations, rituximab, plasma exchange, corticosteroids, and rarely cyclophosphamide. The risk of glomerulonephritis appears to be reduced following SVR [START_REF] El-Serag | The effects of sustained virological response to direct-acting anti-viral therapy on the risk of extrahepatic manifestations of hepatitis C infection[END_REF][START_REF] Fabrizi | Direct-Acting Antiviral Agents for HCV-Associated Glomerular Disease and the Current Evidence[END_REF].

Most HCV-CryoVas manifestations have been shown to resolve after DAA therapy. Across all published DAA studies in patients with symptomatic HCV-CryoVas, longterm follow-up (up to 24 months) has shown high rates of clinical response, either complete (65%) or partial (20%) (Figure 5). Viral clearance was strongly associated with response of vasculitis manifestations. However, some symptoms of CryoVas seem to be more prone to resolve after SVR while others may remain. On one hand, cutaneous manifestations such as purpura and skin ulcers, articular manifestations and myalgia were most frequently reported to respond rapidly, usually with complete resolution (Figure 5A). On the other hand, symptoms related to peripheral neuropathy and renal disorders showed a less frequent complete response compared with cutaneous and articular manifestations (30%-70% versus 75%-100%, respectively) (57,60-66).

Consistent with international guidelines, anti-viral therapy should be started without delay in HCV patients with CryoVas manifestations, with an absolute need for the use of DAA therapy to eliminate HCV infection [START_REF] Saadoun | Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation[END_REF][START_REF] Boyer | CD4+CD25+ regulatory T-cell deficiency in patients with hepatitis C-mixed cryoglobulinemia vasculitis[END_REF]. A combination of DAA with plasmapheresis (to clear the cryoglobulins) and/or rituximab (to deplete cryoglobulinproducing B cells) is tailored according to the severity of CryoVas. Patients with mild to moderate CryoVas symptoms (fatigue, arthralgia, purpura, sensory neuropathy) should be treated only with optimized DAA therapy. Severe CryoVas manifestations, such as membranoproliferative glomerulonephritis, skin necrosis, severe neuropathy, gastrointestinal vasculitis, lung involvement, and heart and/or CNS vasculitis, require additional immunosuppressive strategies in combination with DAA therapy [START_REF] Galli | Recommendations for managing the manifestations of severe and life-threatening mixed cryoglobulinemia syndrome[END_REF]. Anti-CD20 monoclonal antibody (rituximab) is the first choice, most frequently administered at a dosage of 375 mg/m² every week for 4 consecutive weeks, and should always be associated with DAA therapy, although not necessarily at the same time [START_REF] Ramos-Casals | Evidence-based recommendations on the management of extrahepatic manifestations of chronic hepatitis C virus infection[END_REF][START_REF] Ferri | Treatment with rituximab in patients with mixed cryoglobulinemia syndrome: results of multicenter cohort study and review of the literature[END_REF](71).

Removal of circulating cryoglobulins by therapeutic plasmapheresis is accepted as an adjunctive therapy for fulminant cases or severe exacerbation of vasculitis, especially in those with rapidly progressive glomerulonephritis, central nervous system involvement, cardiac manifestations, ulcers or cutaneous necrotizing vasculitis [START_REF] Desbois | Cryoglobulinemia vasculitis: how to handle[END_REF][START_REF] Zignego | Expert opinion on managing chronic HCV in patients with mixed cryoglobulinaemia vasculitis[END_REF]. Of note during plasmapheresis is that the exchange solution should be warmed to body temperature to avoid cryoglobulin precipitation. In case of acute cryoglobulinemic symptoms, 3 to 8 plasmapheresis procedures should be considered every 1 to 3 days with a 1-1.5 total plasma volume [START_REF] Bonacci | Long-Term Outcomes of Patients With HCV-Associated Cryoglobulinemic Vasculitis After Virologic Cure[END_REF][START_REF] Rockx | Plasma exchange for treating cryoglobulinemia: a descriptive analysis[END_REF]. Alternative therapies are limited. Low-dose corticosteroids may help to control inflammatory symptoms such as joint pain. Other conventional immunosuppressants should be given only in case of severe and refractory forms of CryoVas, which are frequently associated with underlying B-NHL [START_REF] Saadoun | Using rituximab plus fludarabine and cyclophosphamide as a treatment for refractory mixed cryoglobulinemia associated with lymphoma[END_REF]. After HCV clearance following DAA therapy in those with a partial response of CryoVas manifestations despite first-line immunosuppressive therapy, the persistence of cryoglobulinemia or relapse of vasculitis symptoms need to be monitored and alternative therapies assessed (low-dose interleukin 2, belimumab) [START_REF] Saadoun | Regulatory Tcell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF][START_REF] Ostojic | Managing refractory cryoglobulinemic vasculitis: challenges and solutions[END_REF].

The safety and efficacy of DAA regimens are excellent in patients with HCV-CryoVas.

Less than 5% of patients discontinue DAA treatment [START_REF] Comarmond | Treatment of chronic hepatitis C-associated cryoglobulinemia vasculitis at the era of direct-acting antivirals[END_REF]. Of note is that in the DAA era the mortality rate of HCV-CryoVas is less than 3% compared with 40 to 60% in the IFN era [START_REF] Comarmond | Treatment of chronic hepatitis C-associated cryoglobulinemia vasculitis at the era of direct-acting antivirals[END_REF]. However, achievement of an SVR is necessary but not sufficient to achieve a complete immunological response, as 6 to 12 months following DAA therapy cryoglobulins remain present in about 50% of patients (Figure 5B). These patients remain at risk of vasculitis relapse and/or of developing B-NHL, and they should benefit from long-term follow-up.

HCV-related lymphoma

The different benefits of SVR in NHL have been known since the IFN era. As mentioned above, a large historical cohort showed evidence of a clear reduction in the incidence of B-NHL in patients with an IFN-induced SVR (HR = 0.7) but not for DAA due to several potential confounders [START_REF] Ioannou | Eradication of Hepatitis C Virus Is Associated With Reduction in Hematologic Malignancies: Major Differences Between Interferon and Direct-Acting Antivirals[END_REF]. Another large retrospective cohort of American veterans found similar biases when evaluating the potential benefit of DAA therapy in NHL risk [START_REF] El-Serag | The effects of sustained virological response to direct-acting anti-viral therapy on the risk of extrahepatic manifestations of hepatitis C infection[END_REF]. Concerning the impact in B-NHL progression, the proven effect of IFN also seems reproducible with DAA.

In international guidelines, HCV treatment is prioritized for those with clinically significant extrahepatic manifestations, including B-NHL [START_REF] Torres | The oncologic burden of hepatitis C virus infection: A clinical perspective[END_REF][START_REF] Zignego | International therapeutic guidelines for patients with HCV-related extrahepatic disorders. A multidisciplinary expert statement[END_REF]. A recent metaanalysis comprising 58 studies reinforced the correlation between SVR and progressionfree survival (PFS), regardless of the type of antiviral therapy used [START_REF] Masarone | Hepatitis C virus infection and non-hepatocellular malignancies in the DAA era: A systematic review and meta-analysis[END_REF]. Despite the small sample available for DAA-induced SVR (n = 76 patients), its pooled data yielded an OR of 8.97 (95% CI 2.27-35.37) for reaching PFS. Studies evaluating B-NHL response while on DAA treatment and complete and partial responses are shown in Figure 6. Both low-and high-grade B-NHL appear to respond to DAA, either alone or in combination with chemotherapy, with a 1-year PFS ranging from 75% to 100%.

Small lymphocytic lymphoma (also known as chronic lymphocytic leukemia) seems to be the only exception among B-NHL subtypes [START_REF] Arcaini | Interferon-free antiviral treatment in B-cell lymphoproliferative disorders associated with hepatitis C virus infection[END_REF][START_REF] Frigeni | Interferon-free compared to interferon-based antiviral regimens as first-line therapy for B-cell lymphoproliferative disorders associated with hepatitis C virus infection[END_REF]. To date, the largest cohort evaluated 66 patients (MZL = 53), with 68% of them responding to isolated DAA therapy [START_REF] Frigeni | Interferon-free compared to interferon-based antiviral regimens as first-line therapy for B-cell lymphoproliferative disorders associated with hepatitis C virus infection[END_REF]. In patients with low-grade B-NHL, HCV treatment has been recommended as first-line therapy without chemotherapy [START_REF] Torres | The oncologic burden of hepatitis C virus infection: A clinical perspective[END_REF][START_REF] Zignego | International therapeutic guidelines for patients with HCV-related extrahepatic disorders. A multidisciplinary expert statement[END_REF]. Regarding high-grade B-NHL, a retrospective cohort evaluated DAA combined with chemotherapy in 47 patients and reported a complete hematological response rate of 98% and a 2-year PFS of 93.1% [START_REF] Merli | Direct-Acting Antivirals in Hepatitis C Virus-Associated Diffuse Large B-cell Lymphomas[END_REF]. Mild non-hepatic adverse events were reported in 11 (23%) patients, while liver toxicity was experienced by 23 patients (49%), mainly in those receiving DAA after chemotherapy. In turn, another study that evaluated 20 DLBCL patients receiving concomitant DAA and chemotherapy did not find differences in the adverse event rate when compared with a historical cohort receiving only chemotherapy [START_REF] Persico | Efficacy and safety of new direct antiviral agents in hepatitis C virus-infected patients with diffuse large B-cell non-Hodgkin's lymphoma[END_REF].

Although the optimal management of high-grade B-NHL in HCV patients has yet to be determined, prior administration of DAA should be able to normalize liver enzymes before chemotherapy, while the concomitant approach achieves this normalization earlier. Considering potential drug-drug interactions and other clinical particularities, these patients should be treated with an individualized and multidisciplinary approach, along with close monitoring of both therapies (89).

Conclusion

Three decades after first being detected, HCV has been shown to induce many B-cell lymphoproliferative disorders ranging from hypergammaglobulinemia and mixed cryoglobulinemia to MZL and B-NHL. This is supported by substantial epidemiological, pathophysiological and therapeutic evidence. The exact pathogenic pathway that leads from one extreme to another is not fully understood, especially with regard to the presence of the antigen stimuli up to the last steps ultimately leading to overt malignancy. This has direct implications in treatment strategies. While the role of DAA has been well established in CryoVas patients, its positive impact on B-cell NHL needs to be confirmed in larger studies with longer follow-up.

Figures Figure 1. Main HCV-cryoglobulinemic vasculitis manifestations

The target organs of cryoglobulinemic vasculitis are illustrated together with the respective frequencies with which they occur. Basic epidemiological aspects are shown in the upper left corner. The first steps of HCV-related lymphoproliferation are induced by chronic antigen stimulation. E2 protein binds to B cells via a membrane protein complex that includes the CD81 receptor. This binding reduces the B-cell threshold for activation and proliferation, and induces immunoglobulin somatic hypermutation, resulting in hypergammaglobulinemia. Also, E2-CD81 interaction leads to bcl-2 rearrangement or t(14;18) translocation, which are known apoptosis inhibitors. After many years of such stimulation, probably associated with other factors (regulatory T cell deficit, bcl-2, cytokines), some abnormal immunoglobulins named cryoglobulins are produced: first type III (polyclonal) then type II (polyclonal with monoclonal component) mixed cryoglobulinemia. In some patients, these cryoglobulins will induce symptomatic cryoglobulinemia vasculitis. The presence of the monoclonal component in type II cryoglobulinemia demonstrates the underlying B cell clonal proliferation. While this process remains exclusively dependent on viral stimulation, it can be reversed after achieving sustained clearance of HCV. However, this B-cell clonal expansion may turn out to be antigen-insensitive in some patients, leading to uncontrolled B-cell proliferation, which is the definition of B-cell lymphoma. Indeed, it is postulated that clonal B cells might acquire their own autonomy throughout the lymphoproliferation process. This is at least partially explained by the "hit and run" theory, also supported by several other factors, such as cytokines, mutations in proto-oncogenes and tumor suppressor genes, oxidative stress and finally dysregulation in intracellular networks.

Underpinning the lymphoproliferative continuum that exists in chronic HCV infection, cryoglobulinemic vasculitis is by far the greatest risk factor for lymphomas. High-grade lymphomas (e.g., DLBCL) typically arise from the transformation of low-grade lymphomas (e.g., MZL). However, high-grade lymphomas can also occur as de novo, that is, without necessarily passing through the low-grade lymphoma stage. Follicular subtype appears to follow a distinct course from MZL and de novo DLBCL, which is reviewed in more detail by Couronné et al [START_REF] Couronné | From hepatitis C virus infection to B-cell lymphoma[END_REF]. AID: activation-induced cytidine deaminase; BLyS: B-lymphocyte stimulating factor; DLBCL: diffuse large B-cell lymphoma; HCV: hepatitis C virus; Ig: immunoglobulin; IL-6: interleukin-6; MZL: marginal zone lymphoma; NO: nitric oxide.

  urinalysis alterations are frequently seen, although it often occurs without clinical repercussion. The more frequent and severe clinically relevant renal manifestation is membranoproliferative glomerulonephritis. Acute renal failure and nephritis syndrome may be the first manifestations of renal involvement. Renal biopsy shows type I membranoproliferative glomerulonephritis in more than 70% of patients. Less frequent renal histopathologic patterns include mesangio-proliferative or membranous glomerulopathy. Other associated histopathological lesions reported include the presence of crescents, necrotizing vasculitis, interstitial inflammation and capillary luminal cryoglobulin thrombi. Immunofluorescence studies demonstrate IgG, IgM, and C3 within most of the cryoglobulin deposits present in the capillary loops; C1q is seen in about one-third of patients. Histological features of the nerve and kidney in CryoVas are shown in Figure 2.

Figure 2 .

 2 Figure 2. HCV-cryoglobulinemic vasculitis pathology

Figure 3 .Figure 4 :

 34 Figure 3. Immune responses leading to autoimmunity during HCV infection

Figure 5 .

 5 Figure 5. HCV-related cryoglobulinemia vasculitis response to DAA therapy.

Figure 6 .

 6 Figure 6. B-cell non-Hodgkin lymphoma hematological response to DAA therapy.

Conflict of interest statement:

CryoVas. B: immunological response (i.e., circulating cryoglobulin clearance) in CryoVas patients. * Total skin manifestations (i.e., both purpura and ulcers) NA = not available