Evolution of Cryoglobulinemia in Direct-Acting Antiviral-Treated Asian Hepatitis C Patients With Sustained Virological Responses: A 4-Year Prospective Cohort Study

Background How cryoglobulinemia evolves after sustained virological response (SVR) following direct-acting antiviral (DAA) treatment in Asian hepatitis C virus (HCV)-infected patients remains elusive. Methods A prospective cohort study was conducted in 422 Taiwanese patients (358 completed DAA therapy and 353 experienced SVRs). Serum cryoglobulins were surveyed at baseline and every 3-6 months posttherapy. Results Of 422, 227 (53.8%) had cryoglobulinemia, 8 (1.89%) had cryoglobulinemic vasculitis. Of 227, 54 (23.8%), 57 (25.1%) and 116 (51.1%) had 1, 2 and 3 cryoglobulins, respectively; those with 3 cryoglobulins had the highest alanine aminotransferase, immunoglobulin G (IgG) and fibrosis-4 index. During a 4-year follow-up, among SVR patients, cryoglobulinemia rates decreased from 56.4% to 15.4%, single cryoglobulin rates increased (21.6% to 63.9%) and 3 cryoglobulin rates decreased (55.7% to 11.1%). Compared with baseline values, among SVR patients with baseline cryoglobulinemia, complement component 4 levels increased, and IgG and IgM levels decreased until 48 weeks posttherapy for those without posttherapy cryoglobulinemia. All 8 cryoglobulinemic vasculitis patients exhibited SVRs; 5 (62.5%) achieved complete clinical response 12 weeks posttherapy, of whom, 2 (40%) experienced clinical relapse 24~48 weeks posttherapy. Baseline IgM levels were associated with posttherapy cryoglobulinemia in SVR patients (cut-off values at 12, 24, 48 weeks and 4 years posttherapy: 130, 105, 118 and 168 mg/dL, respectively). Conclusions Among DAA-treated SVR patients, in 4 years, cryoglobulinemia rates decreased from 56.4% to 15.4%, multiple cryoglobulin rates decreased, cryoglobulinemia signals reversed, 62.5% of cryoglobulinemic vasculitis patients achieved complete clinical response (40% had relapse), and baseline IgM levels indicated posttherapy cryoglobulinemia.


INTRODUCTION
Hepatitis C virus (HCV) is a human pathogen responsible for acute and chronic liver disease. It is chronically infecting an estimated 71.1 million individuals worldwide (1) and classified into 8 genotypes (2). In addition to hepatic complications such as cirrhosis and hepatocellular carcinoma, HCV causes many extrahepatic complications, including mixed cryoglobulinemia (3), obesity, diabetes and dyslipidemia (4). Mixed cryoglobulinemia is the most common HCV-associated extrahepatic complication; up to 90% of patients with mixed cryoglobulinemia harbor HCV infection, and over 60% of patients with chronic HCV infection (CHC) have circulating mixed cryoglobulins (3,5). Most of the information regarding HCV-associated mixed cryoglobulinemia came from studies conducted in Europe (6)(7)(8) or the USA (9). By contrast, in Asia, mixed cryoglobulinemia is an elusive condition. We previously showed that over 60% of Taiwanese patients with CHC had mixed cryoglobulinemia (3), and even among patients with spontaneous HCV clearance, approximately 1/3 had mixed cryoglobulinemia (10). Mixed cryoglobulinemia thus is not a minor issue for Asian individuals with HCV infection. Up to 8 years posttherapy, the prevalence of mixed cryoglobulinemia among CHC patients who achieved sustained virological responses (SVRs) following interferon-based anti-HCV therapy was still 17.64% (3). As interferon has immune-modulating functions, the persistence of mixed cryoglobulinemia after SVR may be induced by the withdrawal of interferon-based therapy (11). With the advent of direct-acting antivirals (DAAs), which target specific HCV proteins during its life cycle (12), anti-HCV treatment has resulted in a high cure rate with a short treatment duration in CHC patients, and the recovery pattern of HCVassociated mixed cryoglobulinemia may not be masked by immune-modulating effects. Several studies in Europe (13)(14)(15)(16)(17), Canada (18) and the USA (19) have shown the beneficial effects of DAA in treating HCV-associated mixed cryoglobulinemia, with complete clinical response rates ranging from 61 to 100%. Although some sporadic cases were reported in Japan (20)(21)(22), the precise effect of DAA in treating mixed cryoglobulinemia has mostly remained unidentified among CHC patients in Asia.
Accordingly, we sought to elucidate this effect in Taiwan, an Asian country where HCV infection is rampant (23), by conducting a 4-year prospective study analyzing serum cryoglobulins of CHC patients before and every 12-24 weeks after the completion of DAA therapy.

METHODS/MATERIALS Patients
The study included subjects aged 18 years or older with CHC who had been surveyed for baseline cryoglobulinemia. Subjects with human immunodeficiency virus or hepatitis B virus infection, hemochromatosis, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, immunosuppressive therapy for any reason, alcoholic liver disease or malignancy and recipients of solid organ transplants were excluded. CHC was defined as detectable HCV RNA for >24 weeks. Cryoglobulinemia was defined as positive for serum cryoglobulin, and cryoglobulinemic vasculitis was defined as described previously (3,19).

Study Design
A total of 422 CHC patients who had been surveyed for serum cryoglobulins were consecutively recruited at a tertiary referral center between May 2015 and December 2019. Of the 422 patients, 358 completed a course of anti-HCV therapy with various DAA combinations (Supplementary Table 1) according to the reimbursement policy of the Bureau of National Health Insurance of the country and were followed for ≥ 12 weeks after completion of DAA therapy. Several baseline factors, including sex, age, body mass index (BMI), HCV RNA, HCV genotype, singlenucleotide polymorphisms of interferon-l3-rs12979860, cryoglobulin, estimated glomerular filtration rate (eGFR), total cholesterol (TC), triglycerides (TG), homeostatic model assessment for insulin resistance (HOMA-IR) [fasting insulin (mU/ mL) × fasting glucose (mmol/L)/22.5], alanine aminotransferase (ALT), rheumatoid factor (RF), immunoglobulin G (IgG), IgM, complement component 3 (C3), C4 and fibrosis-4 (FIB-4) index [(age (years) × aspartate transaminase (U/L)/(platelets (10 9 /L) × (√(ALT (U/L))], and the presence of hepatic cirrhosis were surveyed and recorded for enrolled patients. Biochemical tests were performed at the clinical pathology laboratories of the hospital using routine automated techniques, serum cryoglobulins were measured using the double immunodiffusion method (24), hepatic cirrhosis was screened by abdominal sonography and confirmed by FibroScan. An SVR was defined as undetectable levels of HCV RNA 12 weeks after the completion of therapy. The clinical response of cryoglobulinemic vasculitis following SVR was considered complete if a patient's Birmingham vasculitis activity score (version 3) was 0 or if all affected organs improved 12 weeks after the end of therapy (17). The cryoglobulins and the aforementioned variables were surveyed among the SVR patients every 12-24 weeks after the completion of therapy.

Statistics
All statistical analyses were performed using the Statistical Package for Social Science (SPSS package version 21, SPSS Inc., Chicago, IL, USA) or MedCalc (MedCalc ver. 12.4, MedCalc Software Corp., Acacialaan, Ostend, Belgium) software. The continuous variables are summarized as the means +/standard deviations (SDs), and the categorical variables are summarized as frequencies and percentages. Data were analyzed by one-way analysis of variance (ANOVA) when comparing variables from multiple groups. Post hoc analyses were performed with the least significant difference multiple comparison test. Multivariate logistic regression models were used to assess the relationships between various dependent and independent factors by adjusting for all independent variables with p values <0.1 in the univariate analyses. Hosmer-Lemeshow tests were performed to survey the goodness of fit for the multivariate logistic regression models. Receiver operating characteristic curve (ROC) analyses were performed to evaluate whether independent variables were significant predictors of the dependent variables. The Youden index was assessed to identify the optimal cut-off values of the independent variables according to the coordinate points of the ROC curves. Longitudinal alterations in profiles within the same SVR patients were analyzed and compared using ANOVA, employing general linear model-repeated measures. Paired t tests or nonparametric tests (Wilcoxon test) were performed for the same variable measured in the same subjects at >2 time periods when Mauchly's sphericity tests yielded p values <0.05 during repeated measures. The 95% confidence intervals for mixed cryoglobulinemia rates were calculated based on the Poisson distribution. Statistical significance was defined at the 5% level based on two-tailed tests.

Institutional Review Board
Written informed consent was obtained from each patient. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local institutional review board.

DISCUSSION
The most compelling results of the current study were as follows: (1) Of 422 CHC patients, 227 (53.2%) had baseline cryoglobulinemia; these patients were more frequently female; had higher FIB-4, IgG and IgM levels and cirrhosis rates; and had lower HCV RNA, TC, eGFR, C3 and C4 levels and platelet counts than patients without cryoglobulinemia. Our previous study showed that the baseline rate of cryoglobulinemia in CHC patients was 64.5% (3), which was higher than that (53.2%) in the current study, in which 65 patients had undergone unsuccessful interferon-based therapy, and only 46.5% of the 65 patients had baseline cryoglobulinemia. This finding is consistent with the fact that mixed cryoglobulinemia was linked with a favorable interferon therapeutic response (25), and a lower cryoglobulinemia rate in patients with previous interferon treatment failure (46.5 vs. 64.5%) might reduce the average cryoglobulinemia rate in the current cohort. Compared with patients without cryoglobulinemia, patients with cryoglobulinemia had higher cirrhosis rates and FIB-4 indexes and lower TC levels and platelet counts, indicating that mixed cryoglobulinemia was associated with cirrhosis and poor cholesterol synthesis (3,10), while patients with cryoglobulinemia had higher levels of IgG and IgM and lower levels of C3, C4 and HCV RNA, reflecting the paramount phenotype of mixed cryoglobulinemia (3,10,26). Thus, the baseline comparisons strengthen the reliability of our data.
Interestingly, approximately 1/4 (23.8%) and 1/2 (51.1%) of the patients with cryoglobulinemia had 1 cryoglobulin and 3 cryoglobulins, respectively. The term cryoglobulinemia refers to the presence of one (type I) or more immunoglobulins (types II and III) in the serum (27). Specifically, type I cryoglobulins consist of a single monoclonal isotype. Type II cryoglobulins consist of monoclonal IgM/polyclonal IgG immune complexes, whereas in type III cryoglobulins, both IgM and IgG are polyclonal (28), and both type II and type III cryoglobulins are mixed cryoglobulins. Compared with patients without cryoglobulinemia, patients with 1 cryoglobulin had less severe hepatic inflammation but worse renal impairment; among those with cryoglobulinemia, patients with 3 cryoglobulins were most likely to have mixed cryoglobulinemia (3,10,26) and had the most severe hepatic fibrosis and impaired hepatic synthesis. In particular, no patients with 1 cryoglobulin harbored IgA cryoglobulin, which lessens the possibility of IgA nephropathy, since there is a close relationship between the presence of IgA in cryoglobulinemia immune complexes and its detection in glomeruli (29). All the above results suggested that mixed cryoglobulinemia might develop from the presence of a single cryoglobulin with renal involvement and then progress to the formation of multiple cryoglobulins with frank hepatic involvement, although patients with only one cryoglobulin did not meet the strict criteria for mixed cryoglobulinemia (27). Consistently, the rates of 1 cryoglobulin increased and those of 3 cryoglobulins decreased over time in SVR patients with cryoglobulinemia, supporting the idea that the number of cryoglobulins is correlated with disease stage and progression. Although the positive rates of cryoglobulinemia decreased over time, the cryoglobulins were still detectable in some SVR patients at 4 years posttherapy; some of these patients even had no baseline cryoglobulinemia. The presence of mixed cryoglobulinemia after viral clearance suggests that HCV antigens are not an unfailing component of cryoprecipitating immune complexes (3). Given that CHC massively disturbs the B-cell compartment and generates many large B-cell clones (30), long-term mixed cryoglobulinemia after SVR suggested that some B cells might have reached an HCV-independent autonomous phase to form pathogenic clones before viral clearance (3). Surprisingly, despite the lack of an immune-modulating effect, DAA seems to have a similar efficacy in eliminating long-term cryoglobulinemia as interferon-based therapy, as the cryoglobulinemia rates decreased from 53.2% in all CHC patients to 15.4% in the SVR patients at 4 years posttherapy in the current study (rate difference: 37.8%) were close to those of patients treated with interferonbased therapy (64.5% in all CHC patients to 22% in SVR patients following interferon-based therapy at 4 posttherapy; rate difference: 42.5%) (3). Moreover, among the 8 patients with baseline cryoglobulinemic vasculitis, 8 (100%) had SVRs, 5 (62.5%) achieved complete clinical response at 12 weeks posttherapy, and 2 of 5 (40%) complete clinical responders experienced relapses between 24 weeks and 3 years posttherapy. Namely, 2 of 8 (25%) SVR patients had cryoglobulinemic vasculitis within 3 years posttherapy. Thus, the efficacy of DAA in treating cryoglobulinemic vasculitis is not superior to that of interferon-based therapy, as the rate for 8-year cryoglobulinemic vasculitis in CHC patients following interferon-based therapy was 23.5% in SVR patients with baseline cryoglobulinemic vasculitis (3). However, we want to stress that although relapses of cryoglobulinemic vasculitis did occur in SVR patients following DAA therapy, the relapse symptoms were milder than baseline symptoms, as the main trigger of cryoglobulinemia, HCV, had been eliminated. This finding was consistent with the notion that most relapses of cryoglobulinemic vasculitis were short-lived and were less severe than initially experienced before antiviral therapy (11). The precise triggering factors for relapses after SVR were not identified but were reported mainly to be infection and cancer (3) and demand further investigation. Moreover, patients with open wounds, such as severe skin ulcers subsequent to cryoglobulinemic vasculitis (Figures 2A, B), were not suitable to receive interferonbased therapy due to the risk of bacterial infection and sepsis subsequent to interferon-related immune modulation (11).
Among SVR patients with baseline cryoglobulinemia, at 12 weeks posttherapy, all signals for cryoglobulinemia had been reversed, as the levels of C3, C4, IgG, IgM and RF (3, 10) all changed in those without 12-week posttherapy cryoglobulinemia, and the reversals persisted for all parameters except for C3 and RF until 48 weeks posttherapy for those without any posttherapy cryoglobulinemia. These findings were consistent with the notions that decreased C4 but not C3 was the unfailing stigma for mixed cryoglobulinemia in untreated CHC patients (31), and RF levels were correlated with the severity of mixed cryoglobulinemia in a dose-dependent manner (3).
Among SVR patients, baseline IgM levels were independently associated with posttherapy cryoglobulinemia until 4 years posttherapy and significantly determined posttherapy cryoglobulinemia in SVR patients with various cut-off values for different posttherapy time points. This was consistent with our previous study based on interferon-based therapy (3) and supported the idea that targeting IgM-positive B cells using rituximab (32) is a feasible strategy for the treatment of HCVassociated cryoglobulinemia, even after achieving an SVR, regardless of anti-HCV regimens used. Interestingly, at 48 weeks and 4 years posttherapy, in addition to baseline IgM, baseline BMI and TC levels were associated with posttherapy cryoglobulinemia, respectively. This finding suggests that the baseline metabolic profile might affect long-term cryoglobulinemia through an unknown mechanism, which demands further investigation.
Overall, during a follow-up of 4 years, the prevalence of cryoglobulinemia decreased from 53.8% in CHC patients to 15.4% in SVR patients following DAA therapy; approximately 1/ 4 of patients with baseline cryoglobulinemia harbored 1 cryoglobulin; and in patients with cryoglobulinemia, the prevalence rates of 1 cryoglobulin and 3 cryoglobulins increased and decreased over time, respectively. Among patients with baseline cryoglobulinemic vasculitis, 100% had SVRs, and 62.5% experienced complete clinical response at 12 weeks posttherapy; however, 40% of the complete clinical responders experienced relapses 24 weeks to 3 years posttherapy. Baseline IgM levels were crucial for the development of posttherapy cryoglobulinemia in SVR patients. These findings provide important concepts to monitor CHC patients with cryoglobulinemia treated with DAA and pave the way to identifying a target for treating cryoglobulinemia in SVR patients.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Chang Chang Memorial Hospital. The patients/ participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
M-LC, study design and implementation, manuscript drafting, and critical revision of the manuscript for important intellectual content. J-SC, statistics and manuscript writing. Y-HC, L-HP, T-SW, S-CC, and M-YC, data collection and manuscript writing. R-NC, data collection and manuscript writing and critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. All authors have read the journal's authorship agreement and policy on disclosure of potential conflicts of interest.