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Hepatitis C virus (HCV) represents a major hepatotropic virus, and is responsible of chronic hepatitis leading to cirrhosis and hepatocellular carcinoma. Otherwise, a peculiar tropism of HCV for lymphoid tissue has been clearly demonstrated. This intriguing feature is focused by the pathogenetic role that HCV plays in the so-called "essential" mixed cryoglobulinemia (MC), and also probably in B-cells non Hodgkin's lymphomas (NHLs). In this context, a crucial point is HCV persistence that represents a continuous stimulus for host immune system with consequent B-cell selection and clonal expansion. Autoimmune phenomena like those characterizing mixed cryoglobulinemia are the result of these interactions. The biological effects of cryoprecipitating immune complexes are also mediated by other factors such as complement fractions. In addition, cytokines like CXCL13 (also known as B-cell attracting chemokine-1; BCA-1) play an important role in B-cell homing particularly in intraportal lymphoid aggregates that characterize liver HCV infection. These structures, sometimes resembling secondary lymphoid follicles, may represent the site where B-cell clonal expansions start as antigen-driven events and than expands towards indolent and malignant B cell proliferation.
An intriguing feature stemming from the peculiar tropism of HCV for lymphoid tissue, is the potential evolution into malignant B-cell lymphoma. Though HCV-positive MC should be considered a benign lymphoproliferative disease, in a subset of patients it may precede the development of a malignant lymphoma. Nevertheless, a direct relationship between HCV infection and a subset of NHLs is gaining convincing support. The role of HCV in lymphomagenesis is suggested by several studies; among them, those demonstrating an active HCV replication in lymphocytes and those reported complete or partial remission of NHL in patients achieving a sustained virological response after antiviral therapy, represent a convincing evidence for this correlation. However, a crucial point remains the elucidation of the pathogenetic mechanisms underlying HCV-induced malignant transformation and consequently the definition of a more appropriate therapeutic approach.
Keywords: hepatitis C virus, mixed cryoglobulinemia, non-Hodgkin's lymphoma.â€ƒ
Hepatitis C virus (HCV) is a Flaviviridae family member, genus Hepacivirus, infecting about 200 million people worldwide . About 80% of HCV-infected patients develop chronic hepatitis. Among them, 10-20% evolve into cirrhosis, while 1-5% of cirrhotic patients display an hepatocarcinoma . HCV genome is about 9,600 kb length and encodes for a single protein from an open reading frame of over 9024 nucleotides. This single polyprotein is subsequently cleaved into several structural and non-structural proteins. The structural proteins are represented by core and two envelope proteins (E1 and E2), starting from the 5' end . The ion channel protein p7 derives from E2 cleavage  and is followed by the six non-structural proteins namely NS2, NS3, NS4A, NS4B, NS5A, NS5B. During the replicative stage, HCV genomic RNA is transcribed into a complementary RNA strand. This "negative" strand constitutes a template for a new genomic synthesis and its identification represents a convincing evidence of active replication . Viral proteins are the result of a co- and post-translational cleavage of a single polyprotein, while host peptidases catalyze the cleavage of structural proteins. HCV particles form a membrane-associated replication complex; after genome amplification and protein expression, progeny virions are assembled and released [5, 6].
Although HCV is primarily hepatotropic, its clinical feature is characterized by the emergence of several extrahepatic manifestations. After the identification of HCV as the etiologic agent of non-A, non-B chronic hepatitis and the availability of a serologic test for the demonstration of IgG anti-HCV in the early 90's, several Authors described an intriguing association between HCV infection and "essential" mixed cryoglobulinemia (MC), an immune complex-mediated vasculitis involving small vessels, apart from some geographical differences [7-9]. This association was subsequently confirmed by detection of viral genome in sera of cryoglobulinemic patients with a selective concentration in cryoprecipitates [10, 11]. Incidence of HCV infection in MC ranges from 40 to 90% . Otherwise, HCV-negative MC accounts for about 5-10% .
Since B cell clonal expansion is hallmark of MC , B cell malignant evolution may reflect the occurrence of additional genetic accidents .
Here, we will discuss the currently accepted pathogenetic mechanisms that characterize cryoglobulinemic vasculitis with its peculiar clinical manifestations, the molecular events proposed to explain the potentially malignant evolution, the many clinical features and the currently available therapeutic options for the treatment of MC and B-cell NHLs.
MIXED CRYOGLOBULINEMIA: CLINICAL FEATURES
Cryoglobulins are immunoglobulins (Igs) characterized by insolubility at low temperature (below 37Â°C) and redissolving after warming. In 1933 Wintrobe and Buell first described the phenomenon of the cryoprecipitation in the serum of a patient with multiple myeloma  even if the term "cryoglobulin" was introduced by Lerner and Watson in 1947 . Meltzer et al in a study including 29 patients associated cryoglobulin production to a clinical picture characterized by purpura, weakness and arthralgias that represent a typical symptomatologic triad. These cases were also characterized by increased serum levels of rheumatoid factor (RF) and/or organ dysfunction .
According to Brouet's classification based on their immunochemical composition, cryoglobulins are defined as single (type I) or mixed (type II and III) . Type I cryoglobulinemia consist of a monoclonal Ig, more frequently of IgM or IgG isotype. IgM cryoglobulins occurs in almost 6% of malignant IgM paraproteinemias, whereas IgG cryoglobulins characterize almost 2% of all myelomas. Type I IgA cryoglobulins are rare . Type II MC accounts for 50-60% of all cryoglobulins. It comprises an IgM monoclonal component, frequently mounting light k chains, and polyclonal IgG. IgM molecules displays a rheumatoid factor activity capable of reacting with intact IgG and/or its F(ab)2' fragment . No monoclonal component is contained in type III MC that accounts for 30-40% of cryoglobulins. Some authors have noted that type III MC may represent a transition form evolving into type II MC .
Mixed cryoglobulins are potentially present in the course of connective tissue and autoimmune diseases, and chronic infections [23, 24]. The term "essential" defines cryoglobulinemic syndromes without an underlying identifiable disease. It is now accepted that the majority of them occurs in HCV chronically infected patients  as the result of specific interactions between the virus and the host immune system .
Initially considered as "essential", mixed cryoglobulinemia (MC) is now recognized as the most common HCV-related extrahepatic disease. It has been estimated that 40-60% of chronically HCV-infected patients produce cryoglobulins, but only15-20% develop full-blown clinical features characterizing MC .
The prevalence of MC shows great heterogeneity according to geographic distribution. It seems to be more common in Southern Europe and in the Mediterranean basin respect Northern Europe and Northern America . Although MC is considered to be a rare disorder with an estimated prevalence of approximately of 1:100.000 (with F:M ratio of 3:1), its true prevalence is unknown because of a misunderstanding of the clinical symptoms that leads the patients to refer to several specialists.
Cryoglobulinemic syndrome can be considered an immuno-mediated systemic vasculitis, involving preferentially small and medium size vessels. Although the classical Meltzer's symptomatologic triad purpura, weakness and arthralgias conventionally represents the peculiar picture, the clinical spectrum of cryoglobulinemic vasculitis varies in relation to the different organ localization.
Cutaneous manifestations represent the most typical clinical sign of cryoglobulinemic vasculitis; they range from palpable purpura of lower limbs to chronic torpid cutaneous ulcers typically located in the supramalleolar regions. Usually, purpura has a clinical course characterized by several recurrent flares that can spontaneously recover with a characteristic brown pigmentation due to hemosiderin deposits as a reliquate. Less frequently purpura can extend to the abdomen, upper limbs and thorax. Although the typical skin lesions are represented by small petechial lesions, other manifestations have been described including Raynaud's phenomenon, livedo reticularis, urticaria and edema. Cutaneous manifestations are often complicated by the occurrence of chronic leg ulcers, that have little or no tendency to heal spontaneously causing pain ad severe discomfort to the patient (figure 1) .
Figure 1. Clinical manifestation of cryoglobulinemic vasculitis. (A) Typical purpuric lesions of the legs that sometimes appear confluent (B). (C) and (D): livedo reticularis. (E) chronic leg ulcers. (F) ulcers healing after therapy
The histopathologic feature of palpable purpura is characterized by a non-specific inflammatory infiltrate involving small vessels (leukocytoclastic vasculitis); mononuclear cells may infiltrate vessels' walls. Sometimes endoluminal thrombi and fibrinoid necrosis of the arteriolar walls may be described .
As previously reported, skin lesions are almost invariable associated with arthralgias especially involving symmetrically the hands and knees. Weakness is nearly always present.
Kidney involvement represents a common feature of an immune complexes-mediated systemic vasculitis. Renal injury may complicate MC in almost 30% of cases, being present at the diagnosis in 20% of them [12, 30, 31]. In about 50% of cases renal failure have an indolent course, whereas nephritic (14%) or nephrotic (21%) syndrome occur in 14% and 21% of cases, respectively . The most common clinical features are hypertension, proteinuria, microhematuria, red blood cell casts; a defined picture of cryoglobulinemic glomerulonephritis evolve into chronic renal failure in 14% of cases after a mean follow-up of 6 years .
Although kidney involvement is a common feature of systemic vasculitis, cryoglobulinemic nephropathy is considered as a distinct clinical and pathological entity and the etiological role of HCV has been extensively investigated . Type I membranoproliferative glomerulonephritis is predominantly associated with HCV-infection [35, 36]. The light microscopy observation of bioptic samples shows a pronounced glomerular lobulation with a diffuse thickening of glomerular capillary wall and monocytes infiltration. PAS-positive hyaline thrombi may be observed inside capillary lumina [35, 37]. The mechanism of HCV-induced renal damage is unclear. HCV core protein resulted homogeneously distributed along the glomerular capillary wall and tubulo-interstitial blood vessels  in association with an anti-core activity, suggesting a major role of these immune complexes in the pathogenesis of renal damage .
The involvement of the nervous system in the course of HCV-related MC ranges from 17% to 60% . Sometimes, peripheral neuropathy can represent the first clinical sign of cryoglobulinemia . Peripheral nervous system involvement may complicate MC as sensory-motor neuropathy especially of the lower limbs characterized by paresthesias with loss of strength, pain and burning sensations . Less frequent is central nervous system involvement, characterized by transient dysarthria, hemiplegia and confusional state .
As HCV infection represents the underlying condition characterizing MC, liver is involved in almost 70% of cases In the majority of cases the clinical picture is characterized by a chronic hepatitis with a histopathologic aspect of chronic active hepatitis that can evolve into cirrhosis and hepatocellular carcinoma [7, 44].
Gastroenteric system and Lung
Less common clinical pictures of cryoglobulinemic vasculitis are represented by gastrointestinal (2-6%) and pulmonary (5%) involvement. Intestinal ischaemia may arise with acute abdominal pain; intestinal perforation is also described as well as symptoms that mimic cholecistitis and/or pancreatitis .
Lung involvement in MC is characterized by interstitial pneumopathy in patients displaying dyspnea and dry cough. An acute alveolar haemorrhage with haemoptysis, respiratory failure, and a radiologic demonstration of multiple infiltrates is rare [46, 47].
Classification of cryoglobulinemic vasculitis
Currently there are no classification criteria commonly accepted for the cryoglobulinemic vasculitis even if a clear diagnosis and staging of the disease represent a crucial factor to establish a more precise clinical and therapeutic approach to this multifaceted disease. Recently the Italian Study Group on Cryoglobulinemia (GISC) has proposed a tentative of preliminary classification criteria for cryoglobulinemic vasculitis . On the bases of other classification systems about autoimmune diseases like Sjögren's syndrome, the preliminary criteria for the classification of cryoglobulinemic vasculitis include different questionnaire, clinical and laboratory parameters. This interesting approach showed a good sentitivity and sensibility but need to be validated.
PATHOGENESIS OF HCV-INDUCED MC
The hypothesis of an infectious agent as the etiologic factor of mixed cryoglobulinemia has been always considered. In particular, since the correlation with chronic hepatitis was reported in the first paper by Meltzer , a possible pathogenetic role of some hepatotropic virus has been suspected for a long time. Levo et al.  suggested a possible role of hepatitis B virus infection but this hypothesis was set aside because HBV viremia was rarely recorded and anti-HBV antibodies largely varied among different MC populations. Currently HBV is considered as a causative agent of MC in about 5% of cases .
In the early 90's, after the identification of HCV as the major etiologic agent of non-A, non-B hepatitis, different Authors demonstrated a high prevalence of anti-HCV antibodies in MC patients [9, 51]. This correlation was subsequently confirmed by the detection of HCV genomic sequences that resulted more concentrated in the cryoprecipitates against the correspondent supernatants . In addition, HCV-related proteins were also demonstrated in liver, skin and renal tissues of MC patients [29, 38] as well as in lymph nodes and in circulating CD34+ hematopoietic progenitor cells [52, 53].
The intrinsic mechanism by which HCV promotes cryoglobulin production remains unclear. Since virus persistence may represent a continuous stimulus for the host immune system that is unable to sinthetyze neutralizing antibodies [54, 55], cryoglobulins may be considered the result of these interactions and the presence of IgM molecules with RF activity a crucial event in the cryoprecipitating process . These IgM molecules are almost always associated with light chain 17.109 and heavy chain G6  cross-idiotypes, considered as the product of a restricted expression of germline genes .
It has been hypothesized that the composition of cryoprecipitating immune complexes (ICs) in the course of chronic HCV infection include IgM-17.109 RF molecules which bind anti-HCV IgG . Among viral antigens, the core protein plays a crucial role in cryoglobulins constitution being the relevant ligand for IgG (figure 2) .
Figure 2. Serum cryoprecipitation at low temperature (+4Â°C) in a Wintrobe's tube. Cryoprecipitating immune complexes are represented by IgM-RF that binds IgG with anti-HCV specificity. Of crucial importance is the role of complement fraction like C1q.
Interaction between HCV and lymphocytes is capable of modulating cell functions; in particular, an in vivo activation and expansion of CD5-positive B cells has been considered the major source of IgM RF molecules in type III MC [58, 59]. Therefore, it has been postulated that an initial activation of these cells may be followed by the emergence of a dominant clone that synthetize a monoclonal RF supporting the development of type II MC after a transition phase in which an IgM clonal heterogeneity may define a type II-type III variant . In a subset of HCV-positive patients with MC, a clonal expansion of IgM+CD27+ B cells expressing hyper-mutated RF-like Ig has been demonstrated in peripheral blood in association to VH 1-69/JH4 and VH 3-20 gene segment restriction . These findings have been interpreted as a B-cell proliferation induced by specific antigen stimulation, thus sustaining the notion that persistent B-cell stimulation may represent a first step to malignant evolution.
A crucial role in the composition of cryoprecipitating ICs is played by complement system. Generally, the result of complement binding to setting up ICs consist of size reduction thus maintaining ICs in solution . It is possible to identify two different compartments in sera of MC patients in which higher levels of C3 and C4 fractions are present in the soluble phase whereas very low amounts are detectable in cryoprecipitates . On the contrary, C1q protein and C1q binding activity result significantly enriched in the cryoprecipitates . These data support the hypothesis that an efficient engagement of C1q protein by cryoglobulins may represent a crucial factor in the pathogenetic pathway of MC (figure 2).
The receptor for the globular domain of C1q protein (gC1q-R) is capable to directly interact with HCV core protein, thus representing an efficient way to affect the host T and B cell immunity. gC1q-R/ HCV core protein interaction has been considered capable of modulate T cell immune response whereas circulating HCV core protein engagement with gC1q-R expressed on the surface of B-lymphocytes may represent a direct way by which the virus can affect host immunity [62-64]. The wide expression of gC1q-R on the surface of both circulating blood immunocytes and endothelial cells may determine a specific binding to HCV core protein-containing ICs.
Recently, it has been demonstrated that MC patients display higher levels of soluble gC1q-R that reflects an higher specific mRNA expression in blood mononuclear cells . It was also demonstrated that, soluble gC1q-R circulates as a complexed form containing both C1q and HCV core protein in two different binding sites of the molecule (Figure 3).
Figure 3. Demonstration of HCV E2 protein by immunofluorescence in neoplastic B-cells of a MALT-like gastric lymphoma (A), of a neoplastic lymph node (B), of a splenic lymphoma (C) and of bone marrow infiltrate (D).
Lower concentrations of C4d protein, a low molecular weight fragment derived from the cleavage of C4 complement fraction following classic complement pathway activation, have been demonstrated in MC patients' sera than in that from chronic HCV carriers or healthy subjects . Otherwise, C4d fragment deposits characterize almost all skin biopsy samples of cryoglobulinemic vasculitis suggesting that low circulating C4d levels may derive from sequestered fragments in the vascular bed.
HCV core protein, in the presence of high levels of circulating gC1q-R, can exacerbate the inflammatory condition by activation of complement cascade thus determining endothelial cell activation starting an in situ inflammatory response. From a biological point of view, clinical response to antiviral therapy is characterized by a significant reduction of soluble gC1q-R associated to increased levels of C4d and lower viral load .
Cryoglobulinemic syndrome (CS) is a systemic vasculitis capable of changing the clinical outcome of HCV-infected patients but its long-term impact on the course of HCV-infection has not been assessed. In 2004 Ferri et al., in a study of 231 patients, reported a significantly lower cumulative ten years survival from time of diagnosis in cryoglobulinemic patients respect age and sex-matched general population . Other factors recognized as associated to a poor prognosis are renal involvement, widespread vasculitis, infectious processes [66, 67].
Recently we completed a prospective study regarding a cohort of 950 chronically HCV-infected patients referring to our Department over a period of about 15 years starting from 1990. MC was found in 246 patients (28%) and 184 of them (74.8%) showed cryoglobulinemic vasculitis. The rate of progression of liver fibrosis was lower in patients with CS than in those without; at the same time the probability of developing cirrhosis and hepatocellular carcinoma resulted higher in patients without CS (24.9% vs. 14.2%, p < 0.005 and 20.3% vs. 7.5%, p = 0.003, respectively). Otherwise, extrahepatic complications like renal failure, neurological impairment or B-cell malignancy evolution were more frequent in patients with CS than in those without (32.6% vs. 3%, p < 0.0001; 31.2% vs. 4.8%, p < 0.0001 and 15% vs. 7.1%, p = 0.003, respectively) (unpublished data; manuscript submitted). However, the 15-years survival rate was similar in HCV-infected patients with or without CS, despite different morbidity features and causes of death (70.2% vs. 71.7%).
In the pre-HCV era, management of MC was conventionally based on the use of corticosteroids and immunosuppressive drugs like cyclophosphamide with the aim to prevent irreversible organ failure, reduce pain and improve patients' quality of life. In 1987 recombinant IFN-Î± was empirically employed in 7 patients with "essential" MC ; with the subsequent demonstration of the pathogenetic role of HCV , IFN-Î± became a rational therapeutic strategy. The introduction of pegylated IFN-Î± and subsequently that of ribavirin, changed the therapeutic scenario of chronic hepatitis C increasing virological responses [69-71]. This combination has been shown to be effective in a remarkable proportion of HCV-related MC patients, resulting in a complete clinical response and sustained virological response (SVR) in 78% of the patients . In addition, serum levels of C3 and C4 complement fractions normalized in 80% and cryoglobulins disappeared in 56% of the patients. Even when the antiviral treatment results in resolution of vasculitis, no or only partial improvement in neuropathy and glomerulonephritis is observed, suggesting that the clinical outcome may be conditioned by factors other than the virus .
Extra-hepatic manifestations of chronic HCV infection like MC are characterized by B cell clonal expansions (including RF-synthetizing B cells) [12, 73-75] that have been demonstrated in at least three different compartments, namely liver, bone marrow and the circulation. Consequently, deletion of B-cell clonalities may provide a further rational way to treat MC. It is well known that CD20 antigen, a transmembrane protein, is selectively expressed on pre-B and mature lymphocytes, and that CD20-positive cells are remarkably expanded and activated in patients with MC [76, 77].
On the basis of the demonstration of the effectiveness of Rituximab (RTX), a chimeric monoclonal antibody specifically directed to CD20 antigen, in autoimmune and lymphoproliferative disorders [78-80], it seemed logical to propose its use in HCV-related MC patients refractory to, or relapsing after, conventional antiviral therapy. RTX resulted effective, safe and well tolerated in MC patients both in those resistant to and in those recurring after previous treatments [81, 82]. On these bases, several subsequent papers have addressed the issue of the use of RTX, alone or in combination with steroids [83, 84].
Since an increased viremia was frequently reported in responsive patients, we proposed a triple therapeutic combination (pIFN-Î± plus RBV plus RTX), designated with the acronym PIRR . 22 HCV-positive MC patients received PIRR therapy, whereas 15 additional patients with the same pathology received, by comparison, pIFN-Î± plus RBV with the exclusion of RTX. Follow-up was protracted for 36 months from the end of treatment. Results showed a complete response in 54.5% of patients treated with PIRR, and only in 33.3% of those who were given pIFN-Î± plus RBV without RTX (p<0.05). Even more interesting were the observations that: a) in the large majority (83â€¢3%) of the responders belonging to the PIRR-treated group, a conversion of B-cell populations from oligoclonal to polyclonal was recorded in the liver, bone marrow and peripheral blood compartments; b) compared with 40% of the control group, in all patients of the PIRR group the CR was maintained throughout the follow-up period.
Whether RTX should be administered to patients with cryoglobulinemic vasculitis as first- or second-line therapy, remains to be established .
Of particular interest is the question about MC patients that do not obtain an SVR or those patients showing a continuous cryoglobulin production despite virus eradication. In the first case the use of the new direct-acting antivirals (DAAs) like Telaprevir or Boceprevir (recently approved by the FDA for the treatment of HCV genotype 1 chronic infection) may represent a further therapeutic option . Persistence of MC vasculitis in patients achieving a SVR represents an emerging picture following antiviral and B-cell depletive combined therapies [88, 89]. In these patients a different immunochemical structure of circulating immune-complexes may be postulated; the use of corticosteroids, cyclophosphamide, RTX or Ofatumomab (an IgG1k fully humanized CD20 MoAb) may be considered .
Therapeutic apheresis is a palliative procedure that can be extremely useful for the treatment of severe, life-threatening vasculitis  as well as for the treatment of chronic leg ulcers in patients resistant to other therapies .
Others additional therapeutic approaches for MC have been proposed, like tyrosine kinase inhibitor imatinib, anti-angiogenic drugs like thalidomide, bortezomib (a proteasome inhibitor) and IL-2, but future controlled studies are required to establish if these agents will improve MC therapy [92, 93].
HCV INFECTION AND MALIGNANT LYMPHOPROLIFERATION
About 15% of all human tumors have a viral origin. In developing Countries the percentage of virus-related cancer is 3-fold higher than in developed areas thus reflecting an higher prevalence of oncogenic viruses infection other than the exposure to enhancing co-factors . The pathogenetic role of some viruses in human tumors has been clearly described. This is the case of Epstein-Barr virus (EBV) etiologically linked to Burkitt's lymphoma and probably other tumors ; the human papillomavirus (HPV) may cause cervical, ano-genital, skin and head and neck cancers ; human T-cell leukemia virus type 1 (HTLV-1) is responsible of adult T-cell leukemia  and human herpes virus type 8 (HHV-8) may be related to Kaposi's sarcoma, primary effusion lymphoma and multicentric Castelman's disease .
Among hepatotropic viruses, the role of both hepatitis B (HBV) and hepatitis C virus (HCV) in the etiopathogenesis of hepatocellular carcinoma (HCC) is well defined even if the process of cancerogenesis HBV and HCV-nduced appears quite different. Otherwise, a causative association between HCV and non-Hodgkin's lymphoma (NHL) has indeed been postulated .
After the first observations about the association between HCV and NHL [96, 97], a large number of studies confirmed this report. In a meta-analysis evaluating 15 studies , a relative risk (RR) of all NHL among HCV-positive patients has been reported to be of 2.5 with 95% confidence interval (CI) considering case control studies and 2.0 (95% CI) in cohort studies. In the early studies a prevalence of extranodal NHLs was described in HCV-positive patients as well as a prevalence of some histotypes like lymphoplasmacytic lymphomas. This aspect may be probably due to the observation of malignant lymphomas in HCV-positive patients affected by other low-grade lymphoproliferative disorders like MC (figure 3) . Currently, there are no clear differences on the association between HCV and major histologic B-cell NHL subtypes like diffuse large B-cell, follicular, marginal zone and chronic lymphocytic leukemia/small lymphocytic lymphoma . Otherwise, lymphoma subtypes which do not originate from germinal center or post-germinal center B-cells such as mantle cell NHL, Burkitt lymphoma, T-cell lymphoma and Hodgkin's lymphoma are not consistently linked to HCV infection . The lack of an association with these pathological entities is in line with the notion that proliferation of specific B cell clones following chronic antigenic stimulation seem to be the mechanism that drives subtypes of NHL.
CLINICAL AND HISTOPATHOLOGICAL FEATURES OF HCV-ASSOCIATED NHLs
As previously stated, a growing number of cliniacal and biologic observations have strongly suggested the possible role of HCV in causing a variety of extrahepatic disorders including dermatologic, hematologic, endocrinologic and autoimmune diseases. Among them, a striking association between HCV infection and MC has been clearly demonstrated. On the other hand, MC can be considered an indolent B-cell lymphoproliferative disorder with potential malignant evolution . When HCV-infected patients were analyzed after a long-term observation, progression to NHL was demonstrated in 5-10% of them. Symptoms to progression are usually mild and include an expanding spectrum of autoimmune phenomena like hemolytic anemia, thrombocytopenia and granulocytopenia [102-104].
However, we can define the existence of two different subsets of HCV-associated B-cell NHLs presenting distinct clinical and pathological features. The first is represented by low-grade NHLs evolving from MC, with possible bone marrow involvement and further evolution into an aggressive phenotype. The second possibility is represented by aggressive NHLs without an underlying MC and with bone marrow involvement [102, 105].
It has been calculated that about 13% of patients affected by B-NHL are HCV-positive  and that about 10% of HCV-associated MC patients will develop a B-NHL over a period of ten years follow-up .
A peculiar feature of HCV-associated lymphomas is the extranodal involvement, particularly of the liver, salivary glands, bone marrow and spleen . The most common histotypes are represented by marginal zone lymphomas, lymphoplasmacytic lymphomas and diffuse large B-cell lymphomas . Splenic marginal zone lymphoma, in particular, seems to have an high prevalence in HCV-infected patients with MC . In addition, follicular lymphoma and mantle cell lymphoma can be also associated to HCV infection , as well as mucosa-associated lymphoid tissue lymphoma (MALT) in which HCV can be detected in about 35% of non-gastric MALT lymphomas .
PATHOGENESIS OF HCV-ASSOCIATED NHLs
HCV is capable of chronically persist, thus representing a continuous stimulus for the host immune system that is the cause of B-cell clonal expansions . Also the monoclonal IgM RF synthesis that characterize MC, is the result of the expression of a single dominant clone emerging on the basis of the persistence of viral stimulus [22, 59]. In this context, the capacity of HCV to directly modulate B and T cells function  can be considered as one of the necessary criteria to define HCV as an etiologic factor in lymphomagenesis.
Among the different binding molecules on cells surface that have been described as possible HCV receptors [113, 114], the most known is CD81, a tetraspanin present on the surface of B-lymphocytes (figure 4) .
Figure 4. Schematic representation of HCV-B cells interactions. CD81 is the most known cellular receptor that binds E2 protein. HCV active replication in B-lymphocytes, in addition to the continuous host immune system stimulation, may lead to a "B-cell dysregulation" that progress to oligo/monoclonal expansions. Clinical evolution into autoimmune disease or monoclonal gammopathy of undetermined significance (MGUS) as well as frank lymphoma, may be conditioned by other factors like viral active replication and/or viral protein interactions and/or cytokines and/or host immune response and/or genetic factors.
Interestingly, higher levels of cell-associated viral load as the result of an enrichment of HCV RNA in circulating lymphocytes, have been demonstrated in cryoglobulinemic patients . This phenomenon can be explained as the result of higher receptor density on cell surface or polymorphism of receptor genes [116-118].
In addition, a direct stimulus for lymphocyte proliferation can be sustained by direct infection and active replication of HCV inside B-cells  and this is another crucial factor for the demonstration of HCV as an oncogenic virus. Being HCV a single strand RNA virus with an RNA-dependent RNA polymerase, the demonstration of HCV RNA minus strand is the only molecular marker of an active viral replication. On the contrary, detection of plus strand RNA may be the result of a possible passive contamination of the cells by circulating virions. A direct correlation between an HCV active infection of B cells and MC has been demonstrated by using a highly specific and sensitive method for minus strand HCV RNA detection . These results leads to the demonstration of the peculiar lymphotropism of HCV, being peripheral blood lymphocytes another HCV productive infection compartment and a circulating reservoir of HCV infection (figure 4) .
A peculiar feature characterizing cryoglobulinemic patients is the demonstration of clonally-expanded, RF-synthetizing B cells . By means of PCR amplification techniques, immunoglobulin variable region (IgV) genomic sequences have been analyzed as molecular marker of B cell progeny, demonstrating an antigen-driven B cell clonal expansion. Heavy and light chain IgV gene analysis result in a high mutation rate as usually occurs when derives from a germinal or post-germinal center origin . Little is known about the possible viral antigens capable of inducing such a clonal expansion and no viral protein seems to be a specific ligand for BCR . All expanded B cell clones that are characterized by somatic hypermutation of IgV genes seems capable of recognize a single epitope thus suggesting that they casually arise from a pool of cells selected for non-self antigens, probably in the course of germinal center reaction . Interestingly, many expanded B clones display a complementarity determining region-3 (CDR-3) resembling CDR-3 of rheumatoid factor (RF CDR-3), suggesting that they derive from autoimmune-oriented precursors with anti-IgG specificity.
B-cells proliferation is characterized by a continuous rearrangement of IgV genes that causes different mutants. VDJ region amplification by PCR define the unique combination of N regions with DH and JH regions that can be considered as a clonal marker of cellular progeny. The application of this method leads to the demonstration that B cell clonal expansions are present in the liver tissue of almost 90% of HCV-positive MC patients if compared with blood and bone marrow compartments . Otherwise, the presence of inflammatory infiltrates of the portal tracts resembling follicle-like structures with a functionally active germinal center, is a peculiar feature in liver biopsies of HCV-chronically infected patients [125, 126]. VDJ pattern obtained from these patients showed oligoclonality or monoclonality thus demonstrating that intrahepatic B cells expansions derived from few or single cells; interestingly, each focus may derive from different B cells with the development of unrelated clones [125, 126].
On the other hand, it has been demonstrated that the occurrence on intrahepatic B cell clonal expansions is almost invariably associated with extrahepatic manifestations like MC, high serum levels of RF activity, monoclonal gammopathy of undetermined significance and B cell malignancy. Sequence analysis of IgH CDR-3 gene segments of intraportal B-cell clonalities revealed a wide range of variations, suggesting that they are also the result of an antigen-driven response . On these bases, it can be inferred that B cell clones start expanding in the liver as the result of an IgH-VDJ upregulated mutational activity, and from here migrates to peripheral blood and bone marrow .
So, if we consider that the liver represents the main target of HCV infection and, at the same time, the main site of inflammation, B cell recruitment and expansion, the identification of the factor(s) that contributes to the establishment and progression of this complex clinical spectrum is of crucial importance. In this context, an interesting field of research is represented by the study of some molecules capable of prolonging B-cell survival. Among them, B-cell Activating Factor (BAFF), a chemokine belonging to the TNF family, seems to play an important role in B-cell survival . The most important effect of BAFF is probably the inhibition of apoptosis in B cells. BAFF expression resulted higher in intraportal lymphoid aggregates and skin tissue of cryoglobulinemic patients. It has been hypothesized that BAFF synthesis starts in inflammation sites like liver and skin and then arise the circle .
One of the most important anti-apoptotic factors is represented by Bcl-2 protein. Its upregulation due to t(14; 18) chromosomal translocation is a peculiar feature of follicular B cell NHLs and has been also described in cryoglobulinemic patients . However, in an our previous study no Bcl-2/IgH amplification was detected in intraportal inflammatory infiltrates isolated by means of laser microdissection of liver biopsy of HCV-positive patients . On these basis, it can be hypothesized that heavy chain Ig genes rearrangement is not associated with Bcl-2/IgH chromosomal translocation in liver compartment. A possible explanation of this discrepancy can be represented by ethnical and/or environmental factors in that Bcl-2/IgH rearrangement is less common in Mediterranean area than in northern Europe . Another hypothesis consider that in non neoplastic conditions like MC Bcl-2/IgH rearrangement could be a transient effect due to persistence of viral infection .
Activation-Induced Cytidine Deaminase (AID) is an enzyme involved in the degradation of pyrimidine nucleotides: it is essential for somatic hypermutation and class switch recombination of immunoglobulin genes in B cells . Some studies have proposed a possible pathogenetic role of AID in B-cell lymphomagenesis, in particular during the initiation and progression of B-NHL because a dysregulation in either of these two processes can determine a chromosomal translocation and/or an aberrant somatic hypermutation that are the two main causes of B-NHL associated genetic accidents [132, 133]. It has been postulated that AID is triggered by HCV core protein in human hepatocytes via NFkB activation (figure 5) .
Figure 5. Detection of activation-induced cytidine deaminase (AID) protein in lymph node of a patient affected by HCV-associated B-cell NHL.
All these data support the epidemiological observations about the association between HCV chronic infection and B-NHL even if further studies are necessary to better evaluate pathogenetic mechanisms and optimize therapeutic approaches.
HCV RELATED NHLs: THERAPEUTIC OPPORTUNITIES
As previously described, HCV chronic infection may represent a truly risk factor not only for the development of "indolent" lymphoproliferative disease like mixed cryoglobulinemia, but also for frank B-cell NHL with a wide geographical variability.
Of consequence, the first consideration is that the induction of a sustained virological response following an effective antiviral therapy with pegylated interferon plus ribavirin should exert a preventive effect on lymphomagenesis in HCV-chronically infected patients . On the other hand, based on the observation of gastric MALT lymphoma regression after eradication of H. pylori infection, we can hypothesize that antiviral therapy could induce a regression of NHL.
The efficacy of antiviral therapy was described by Hermine et al.  in patients with villous lymphocytes splenic lymphoma in that a complete remission was achieved in 8/9 HCV-positive patients. Further studies have confirmed these data. A systematic review by Gisbert et al.  reported that about 75% of HCV-infected patients with a lymphoproliferative disorder achieved a complete remission following antiviral treatment.
Recently, Arcaini et al. have reviewed the role of antiviral therapy in HCV-associated indolent B-cell lymphomas . According WHO classification, indolent lymphomas are poorly symptomatic diseases belonging to low-grade lymphoma . They include follicular lymphoma, small lymphocytic lymphoma, marginal zone lymphomas, splenic marginal zone lymphoma, primary nodal marginal zone lymphoma, extranodal marginal zone lymphoma of mucosa-associated tissue (MALT), and lymphoplasmacytic lymphoma. In almost all cases an hematologic response was obtained following a virological response thus confirming the causative link between HCV and lymphomas . Further studies are necessary to establish the efficacy of novel direct antiviral agents like boceprevir and telaprevir that increase SVR rates in genotype 1 HCV infection and then could also increase hematologic response rate in patient with more resistant infection. On the basis of therapeutic regimens for HCV-associated mixed cryoglobulinemia based on the combination of rituximab and antiviral therapy [85, 140] it is possible to hypothesize a similar approach also in indolent B-cell lymphomas.
On the other hand, treatment of HCV-positive aggressive B-cell lymphomas like diffuse large B-cell and mantle cell lymphomas (DLBCL; MCL) with antiviral therapy appears less effective. A possible explanation of this phenomenon may be related to the antigen-independent phase of the lymphomagenesis, in which the malignant evolution is irrespective to virus persistence. These lymphomas require an adequate chemotherapy with or without rituximab, even if the development of hepatitis flares has been described . However, there are some papers reporting clinical remission of DLBCL  and MCL  after antiviral therapy. On these bases, a combination of antiviral therapy and immune-chemotherapy has been proposed to prevent or treat hepatitis flares . However, the use of interferon plus ribavirin combination therapy usually resulted in an increased hematologic toxicity as previously reported . A more interesting approach consist in sequential schedule consisting in immune-chemotherapy followed by pegylated interferon plus ribavirin. The aim of this approach was to induce an SVR in patients obtaining a complete remission of lymphoma after chemotherapy, in order to prevent hepatitis reactivation and a long-term control of NHL , and result effective and well tolerated.
However, these data need further validation in larger prospective studies.
HCV infection should not be considered only as a major cause of liver disease. There are several different biological compartments involved by this virus. In particular, B lymphocytes represent, as well as hepatocytes, another site in which HCV may actively replicate, thus considering B-cells as a viral reservoir.
Viral persistence represents a continuous stimulus for the host immune system leading to B-cell selection and clonal expansion that becomes evident with the synthesis of autoantibodies like IgM with rheumatoid factor activity (IgM-RF) that characterize MC. This process seems to occur in a microenvironment like intraportal lymphoid follicles as a result of a distinct selection process probably supported by cytokine signaling sustaining B-cells activation and proliferation. The peculiar clinical features of MC are the result of the biological activities of the immune complexes constituted by IgM-RF, anti-HCV IgG and viral antigens as well as complement fractions. Among them a peculiar role is played by C1q and by the globular domain of C1q receptor (gC1q-R) that, in combination with viral proteins like core protein, modulates the ICs deposition in the vascular bed leading to cryoglobulinemic vasculitis. A possible role of viral proteins has been described also for B-cells proliferation.
On these bases, MC may be considered a low-grade, indolent, benign lymphoproliferative disease belonging to an antigen-dependent B-cell clonal expansion with potential evolution into a malignant phenotype. Therefore, HCV-related malignant NHLs may derive from benign lymphoproliferation like MC being usually classified as low grade/indolent lymphomas with potential evolution in more aggressive phenotypes, or may arise directly often involving extranodal sites, with an indolent phenotype resembling MALT lymphomas or as highly aggressive lymphomas.
The definition of HCV as an etiologic agent leads to the consideration that antiviral therapy could be effective both in MC and in NHLs. The combination of antiviral therapy with B-cell depletion may be considered as a standard of care for cryoglobulinemic vasculitis, capable of inducing long-term clinical, immunologic and virologic remission. At the same time, a clinical remission of low-grade NHLs after antiviral treatments has also been described. Of consequence, these results confirm the pathogenetic role of HCV in lymphoproliferative diseases and offers new therapeutic options even if further studies are mandatory. Antiviral therapy may also have a role in high grade NHLs in which the pathogenetic process can be considered antigen-independent. In these cases that requires immune-chemotherapy, antiviral treatment may be useful in preventing hepatitis flares but also in preventing relapse if an SVR is obtained.
In conclusion, HCV infection should be considered a multifaceted disease, with potential malignant evolution not only referred to liver compartment (i.e. hepatocellular carcinoma), but also to other targets like lymphoid tissue. However, there are several further aspects that need to be clarified regarding pathogenetic mechanisms of HCV-induced lymphomagenesis. Of great interest will also be the evaluation of the impact of the new direct antiviral agents as well as the new target-therapies inducing B-cell depletion in the prevention of HCV-related malignancies.