Objective: To evaluate the relevance of serum-free light chain (FLC) assessment in hepatitis C virus (HCV)-related lymphoproliferative disorders, including mixed cryoglobulinemia (MC) and B cell non-Hodgkin lymphoma (B-NHL).
Patients and methods: A total of 59 patients infected with HCV were prospectively followed, including patients without MC (n = 17), with asymptomatic MC (n = 7) and with MC vasculitis (n = 35, 9 of whom had B-NHL). Clinical and biological data were recorded at the time of the initial evaluation and at the end of follow-up. Serum FLC quantitation was carried out using a serum FLC assay.
Results: The mean (SD) serum κ FLC level was higher in patients with asymptomatic MC (27.9 (8.6) mg/litre), MC vasculitis (36.7 (46.2) mg/litre) and B-NHL (51.3 (78.3) mg/litre) than without MC (21.7 (17.6) mg/litre) (p = 0.047, 0.025 and 0.045, respectively). The mean serum FLC ratio was higher in patients with MC vasculitis (2.08 (2.33)) and B-NHL (3.14 (3.49)) than in patients without MC (1.03 (0.26)) (p = 0.008). The rate of abnormal serum FLC ratio (>1.65) correlated with the severity of HCV-related B cell disorder: 0/17 (0%) without MC, 0/7 (0%) asymptomatic MC, 6/26 (23%) MC vasculitis without B-NHL and 4/9 (44%) B-NHL (p = 0.002). Serum κ FLC levels and the serum FLC ratio correlated with the cryoglobulin level (r = 0.32, p<0.001 and r = 0.25, p = 0.002, respectively) and the severity of the B cell disorder (r = 0.26, p = 0.045 and r = 0.41, p = 0.001, respectively). Among patients with an abnormal serum FLC ratio at baseline, the FLC ratio correlated with the virological response to HCV treatment.
Conclusions: In patients infected with HCV, an abnormal serum FLC ratio appears to be a very interesting marker, as it is consistently associated with the presence of MC vasculitis and/or B-NHL. After antiviral therapy, the serum FLC ratio could be used as a surrogate marker of the control of the HCV-related lymphoproliferation.
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Hepatitis C virus (HCV) is associated with B cell disorders, including mixed cryoglobulinemia (MC)1 and B cell non-Hodgkin lymphoma (B-NHL).2 Among patients with HCV-related MC, 5 to 10% will develop symptomatic MC vasculitis.3 Monoclonal B cells producing pathogenic IgM with rheumatoid factor activity can be detected in patients with HCV-associated MC,4 and a significant proportion of monoclonal MC type II can evolve into overt B-NHL. In a recent multicentre Italian study, the estimated risk for lymphoproliferative disease was found to be 35-fold in patients with HCV-related MC compared with the general population.5 In a meta-analysis, the prevalence of HCV infection in patients with B-NHL was found to be approximately 15%.6 As such, MC may be considered a low-grade and benign B cell lymphoproliferation and an independent risk factor for B-NHL in patients infected with HCV.5
Epidemiological studies have demonstrated the association between HCV infection and B-NHL.6–11 Most common histological subtypes of HCV-associated lymphomas are low-grade marginal zone lymphomas,5 8 eg, splenic lymphoma with villous lymphocytes (SLVL) and mucosal-associated lymphoid tissue (MALT) lymphomas,12 and more rarely with lymphoplasmacytic lymphomas and large B cell lymphomas.11 13 14 MC is constantly present in HCV-associated SLVL.15 In such patients, antiviral treatment with interferon (IFN)α and ribavirin was effective, whereas it was ineffective in HCV-negative SLVL patients.15 16 Complete virological response correlates with sustained haematological response, and virological relapse is associated with an SLVL relapse. A reduction in HCV viral load after restarting the antiviral treatment correlates with haematological remission. Overall, these data indicate a strong correlation between serum viral load and tumour burden in HCV-associated SLVL, and support the existence of a causal relationship between HCV chronic antigenic stimulation and the marginal zone lymphomatous process.17 However, in patients infected with HCV, the diagnosis of B-NHL is often difficult, and early biological markers are lacking to detect patients with this disease.
Monoclonal B cell lymphoproliferation is associated with the excessive production of monoclonal immunoglobulins. In several B cell and plasma cell disorders, such as amyloid light chain (AL) amyloidosis, multiple myeloma and light chain deposition disease, more light chains than heavy chains are produced that can be detected in the serum. Serum-free light chain (FLC) assessment has been shown to be a useful laboratory test for the diagnosis and monitoring of patients with B cell and plasma cell disorders.18 Abnormalities in serum FLC and mainly in the serum FLC ratio suggest B cell dysregulation and an evolving clonal process. Thus, potential uses could include the early diagnosis and monitoring of B cell lymphoproliferation in patients with HCV infection.
The aim of this study was to analyse the interest of serum FLC assessment in HCV-related lymphoproliferative disorders.
PATIENTS AND METHODS
The study population consisted of 59 patients with HCV infection (HCV RNA+) who were prospectively followed-up, including 17 patients without MC, 7 with asymptomatic MC and 35 with MC vasculitis, 9 of whom had B-NHL. Serum samples were collected at baseline and at the end of follow-up and then stored at −80°C. Informed consent was obtained from each patient, and the study conformed to the ethical guidelines of the Declaration of Helsinki. Patients who were MC positive were defined as having MC in their serum that was over than 0.05 g/litre on at least on two occasions. Symptomatic MC was defined by serum MC associated with the triad of purpura/arthralgia/asthenia and sometimes with renal or neurological involvement.19 All patients had histologically proven chronic active liver disease.
For each patient, clinical and biological data were recorded at the time of the initial evaluation and at the end of follow-up. Clinical data included age, sex, neurological involvement, cutaneous involvement (Raynaud phenomenon, purpura, livedo, distal ulcers), arthralgia, myalgia and clinical signs of hepatic involvement. Laboratory evaluation included a complete haemogram, a serum chemistry profile and determination of the C3 and C4 fractions of complement, rheumatoid factor and cryoglobulin. A urinalysis was also completed to screen for haematuria, and a 24-h urine collection was performed to quantify daily excretion of protein.
Virological and immunological serum markers
HCV antibodies and RNA were detected as previously described.20 HCV genotyping was performed using a second-generation line probe assay (LiPA; Innogenetics, Brussels, Belgium). Cryoglobulin levels were measured and classified as previously described.20 The immunological evaluation included determination of rheumatoid factor and complement components, using standard methods.
Serum FLC assessment
Serum FLC quantification was carried out using a serum FLC assay (Freelite, The Binding Site, Saint Egrève, France) performed on a BN II Nephelometer (Dade Behring, Marburg, Germany). The FLC quantitation consisted of two separate assays: one to detect free κ and the other to detect free λ light chains. The normal ranges are 3.3 mg/litre to 19.4 mg/litre for κ FLC and 5.7 mg/litre to 26.3 mg/litre for λ FLC, respectively. The normal range for a serum FLC ratio is 0.26–1.65. In randomly selected patients with asymptomatic MC (n = 5), MC vasculitis without B-NHL (n = 5) and MC vasculitis with B-NHL (n = 6), FLC immunoassay was also performed on serum samples after standard warming and after warming at 37°C for 1 h to redissolve the cryoglobulin.
Response to antiviral therapy
Clinical response of MC vasculitis to antiviral treatment was defined by analysing the course of the following main clinical signs: skin involvement (absence of purpura), peripheral neuropathy (clinical and/or electrophysiological improvement at two successive examinations), renal involvement (normalisation of serum creatinine level and disappearance of proteinuria) and absence of arthralgia. A complete clinical response was defined by an improvement in all baseline clinical manifestations. A partial response was defined by an improvement in at least half of the baseline clinical manifestations. All other patients were classified as non-responders. Relapse was defined as the reappearance of clinical signs of vasculitis. A sustained virological response (SVR) was defined by the absence of detectable serum HCV RNA (<12 IU/ml) 6 months after the discontinuation of antiviral treatment; the remaining patients were classified as virological non-responders. A complete immunological response was defined by the absence of serum cryoglobulin, and a partial immunological response was defined by a decrease of 50% in the baseline cryoglobulin level.
Data are presented as mean (SD) for continuous variables and percentage for qualitative variables. The Fisher exact test was used to compare qualitative variables and the non-parametric Mann–Whitney U test to compare continuous variables. Correlation was evaluated using the Spearman test. A p value ⩽0.05 was considered significant. Statistical analyses were performed using StatView (SAS Institute, Cary, North Carolina, USA).
The main characteristics of the 59 patients with chronic active HCV infection (29 men and 30 women, mean (SD) age 57.2 (14.2) years (range 31–82)) are given in table 1.
Serum FLC assessment in patients infected with HCV
The mean serum FLC level at baseline was 31.3 (37.3) mg/litre for κ and 20.7 (11.0) mg/litre for λ, and the mean serum FLC ratio was 1.66 (1.86). The serum FLC level was abnormal in 34 (58%) patients for κ and 13 (22%) for λ, and the serum FLC ratio was abnormal in 10 (17%). Results were similar on serum samples tested after standard warming and after warming at 37°C for 1 h.
The mean serum κ FLC level was significantly higher in patients with asymptomatic MC (27.9 (8.6) mg/litre; p = 0.047), MC vasculitis (36.7 (46.2) mg/litre; p = 0.025) and B-NHL (51.3 (78.3) mg/litre; p = 0.045) than in patients without MC (21.7 (17.6) mg/litre) (fig 1A). The mean serum κ FLC level was significantly higher in patients with than in patients without: MC (35.2 (42.3) vs 21.7 (17.6); p = 0.014), type II MC (38.7 (46.3) vs 21.3 (14.9); p = 0.004) and a κ monoclonal component (39.4 (47.7) vs 21.8 (14.5); p = 0.012).
The mean serum FLC ratio was similar in patients without MC and with asymptomatic MC (1.03 (0.26) vs 1.08 (0.33); p = 0.85). It was significantly higher in patients with MC vasculitis (2.08 (2.33); p = 0.008) and B-NHL (3.14 (3.49); p = 0.008) than in patients without MC (1.03 (0.26)) (fig 1B). The mean serum FLC ratio was significantly higher in patients with than in those without: MC (1.92 (2.16) vs 1.03 (0.26); p = 0.02), type II MC (2.10 (2.36) vs 1.07 (0.25); p = 0.016), a κ monoclonal component (2.18 (2.42) vs 1.04 (0.24); p = 0.002), MC vasculitis (2.08 (2.33) vs 1.04 (0.27); p = 0.004) and B-NHL (3.14 (3.49) vs 1.39 (1.29); p = 0.023).
Patients with abnormal serum FLC ratio (n = 10) compared to patients with normal serum FLC ratio (n = 49) showed higher rates of MC (100% vs 65%; p = 0.049), vasculitis (100% vs 51%; p = 0.004), arthralgias (90% vs 45%; p = 0.01) and B-NHL (40% vs 10%; p = 0.036). No peculiar pattern of vasculitis-related organ involvement was found between patients with an abnormal serum FLC ratio and those with a normal ratio. Mean serum C4 complement components were significantly lower in patients with an abnormal serum FLC ratio rather than a normal ratio (0.07 (0.06) vs 0.16 (0.09); p = 0.045).
Correlation between serum FLC levels and the severity of B cell disorders
An abnormal serum FLC ratio was found in 10/59 patients (17%), all with MC vasculitis, with a continuum in the rate of abnormal FLC ratio according to the severity of HCV-related B cell disorder: 0/17 (0%) patients without MC, 0/7 (0%) with asymptomatic MC, 6/26 (23%) with MC vasculitis without B-NHL and 4/9 (44%) with B-NHL (p = 0.015; test for trend p = 0.002) (fig 2). Serum κ FLC levels and serum FLC ratio correlated with the severity of the B cell disorder according to the presence or absence of MC, MC vasculitis and B-NHL (r = 0.26; p = 0.045 and r = 0.41; p = 0.001, respectively).
Serum κ FLC level correlated with cryoglobulin (r = 0.32; p<0.001), and C4 (r = −0.23; p = 0.02) and IgM levels (r = 0.49; p = 0.001). No correlation was found with serum levels of IgG, γ globulins and rheumatoid factor. Serum λ FLC level was not correlated with cryoglobulin, C3 and C4 levels, IgG, IgM and γ globulins levels, and rheumatoid factor. Serum FLC ratio correlated with cryoglobulin level (r = 0.25; p = 0.002), C4 levels (r = −0.37; p<0.001) and rheumatoid factor (r = 0.39; p = 0.003). No correlation was found with IgG, IgM and γ globulin levels.
After a mean follow-up of 69 (18) months, all but one patient with abnormal serum FLC ratio at baseline (n = 10) showed a decrease in the serum FLC ratio (2.05 (2.14) vs 4.42 (3.43); p = 0.014) (fig 3). Of these 10 patients, 8 exhibited an SVR after HCV treatment, 6 of which normalised the serum FLC ratio. Two patients did not reach an SVR: one responder/relapser patient normalised the serum FLC ratio (1.36 vs 1.73), whereas one non-responder patient increased the serum FLC ratio (8.00 vs 7.38).
In the present study, we found higher levels of serum κ FLC in patients infected with HCV with MC, type II MC and κ monoclonal component. More interestingly, we found a higher serum FLC ratio in patients with type II MC vasculitis and B-NHL. The elevation of serum FLC levels may result from polyclonal B cell activation induced by the virus. In autoimmune disorders, such as systemic lupus erythaematosus,21 rheumatoid arthritis and primary Sjögren syndrome,22 an elevation of serum FLC levels was found and correlated with disease activity. The analysis of serum FLC ratio seems to be more interesting and relevant in the presence of HCV infection. In contrast to the polyclonal B cell activation in many autoimmune disorders, HCV infection may induce monoclonal B cell lymphoproliferation. This results in B cell dysregulation and excessive production of monoclonal immunoglobulins, which can be detected early by analysis of the serum FLC ratio.
Patients with an abnormal serum FLC ratio (>1.65) showed a higher prevalence of MC, MC vasculitis and B-NHL than patients with a normal serum FLC ratio, but without a particular pattern of vasculitis-related organ involvement. An abnormal serum FLC ratio was consistently associated with MC vasculitis and/or B-NHL. In the search for new markers to evaluate the individual risk of B cell lymphoproliferation in patients infected with HCV, an, abnormal serum FLC ratio appears to be a good candidate as it is consistently associated with a B cell lymphoproliferation, either benign (MC vasculitis) and/or malignant (B-NHL).
The serum FLC ratio correlates with the severity of the HCV-related B cell disorder, ie, from asymptomatic MC to B-NHL. The increase of the serum FLC ratio in the follow-up of patients infected with HCV may represent a surrogate marker of the immune response switch from polyclonal to monoclonal activated B cells, and of the development of a B cell lymphoproliferation. Not surprisingly, we found a correlation between serum κ FLC levels/serum FLC ratio and serum levels of cryoglobulin and other surrogate markers of MC (ie, C4 complement component and rheumatoid factor). These findings might be related to the fact that in the setting of HCV infection, MC usually consists of monoclonal IgM with a κ light chain (type II MC) or less frequently polyclonal immunoglobulins (type III MC).19 23 In the present study and in agreement with the previous reports, 81% of patients had type II MC (mainly with κ light chain) and 19% type III MC.
Lastly, we found that among patients with abnormal serum FLC ratio at baseline, the FLC ratio was correlated with the virological response to HCV treatment. Previous studies showed that in patients infected with HCV with B-NHL, a complete virological response correlates with sustained haematological response. Virological relapse was associated with the re-emergence of circulating villous lymphocytes and a splenomegaly.16 17 The parallel evolution of the serum FLC ratio while on antiviral therapy and the virological response are in agreement with these studies and support: (1) the strong correlation found between the serum HCV viral load and tumour burden in HCV-related B cell lymphoproliferation; (2) the existence of a relationship between HCV chronic antigenic stimulation and the lymphomatous process.
In conclusion, an abnormal serum FLC ratio is particularly interesting in patients infected with HCV: (1) it is associated with the presence of MC vasculitis and/or B-NHL; (2) after antiviral therapy, it is a surrogate marker of the control of the HCV-related lymphoproliferation.
Competing interests: None.
Ethics approval: Informed consent was obtained from each patient, and the study conformed to the ethical guidelines of the Declaration of Helsinki.
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