In our prospective chronic HCV patient cohort, we aimed to evaluate changes in the serum levels of cryoproteins and in cryoprotein associated disorders, like cryoglobulinemic vasculitis and/or lymphoma, after successful antiviral therapy. HCV eradication and subsequent disappearance of cryoglobulins in patients treated with IFN and ribavirin [18, 19] or pegylated IFN and ribavirin combination [20, 21] was previously demonstrated. Recently the efficacy of DAA therapy on remission of cryoglobulins and its complications has also been documented, however there are only a few studies with long-term follow-up [22,23,24,25,26]. In the majority of our patients DAA therapy was used. Although we investigated a small patient cohort, but with an exceptionally long follow-up period (more than 12 years) that is the longest evaluation period to our knowledge following SVR in patients with mixed cryoglobulinemia. In our study serum levels of cryoglobulins and type of immunoglobulins were detected. Thus, type II and type III forms could be precisely identified. Type II cryoglobulins with monoclonal IgM content were present in most patients (25 type II vs. 15 type III). A novel finding of the present study is a striking quantitative difference, i.e. more than 10 times higher amount of total cryoprotein content, between type II and type III cryoglobulins. Similar differences in RF IgM activity were detected according to type of cryoglobulinemia as well. This demonstrates that in these cases, B cell stimulation is probably much stronger [10], which might explain why the time needed until the full disappearance of type II cryoproteins was also much longer following the eradication of HCV infection as compared to type III. More than 50% of our patients became cryoglobulin negative within one year after achieving SVR. The initial cryoglobulin levels in these 23 patients, however, were 10 times lower as compared to those, who remained cryoprotein positive at that time point. The vast majority of cases in the positive group had type II cryoproteins (15 out of 17), but even their total cryoprotein levels have already decreased significantly by cc. 90% within the first year. Cryoglobulins disappeared within 4 years after SVR in all type III patients, whereas we still have positive patients with type II cryoglobulins even after 12 years of follow up. C4 results are in line with previous observations, i.e. in the presence of cryoglobulins C4 is significantly lower than the lower limit of normal range and C4 normalizes after SVR parallel to the disappearance of cryoglobulins [27].

Due to the prospective design of this study, we have detected 17 patients with asymptomatic cryoglobulinemia. During the follow-up period, all of them remained asymptomatic. The significantly lower level of cryoglobulins compared to symptomatic patients could at least partially explain the absence of clinical symptoms in this group. Symptomatic cryoglobulinemia (benign vasculitis or NHL) was associated with a 9 times higher total protein content as compared to asymptomatic cryoglobulinemia. Furthermore, type II cryoglobulins were present in more than 80% of the symptomatic patients, whereas in asymptomatic patients type III cryoglobulin was the dominant one.

As expected, in our patients, the typical manifestations of cryoglobulinemic vasculitis were skin purpuras, skin ulcers, glomerulonephritis, arthralgias, polyneuropathy and some rare types, like Raynaud’s phenomenon, myopathy, or Sjögren’s syndrome. Another important observation was the presence of more than one manifestation of cryoglobulinemic vasculitis at the same time in several patients. We also observed immediate improvement of vasculitis after achieving SVR in most cases. In 43% of these patients (10 out of 23), vasculitis disappeared within one year after eradication of HCV. However, in five patients skin purpuras were so severe that immunosuppressive therapy became necessary. In three patients, sensory polyneuropathy and in one Raynaud’s phenomenon were also present. Fortunately, in their cases, with additional therapy, vasculitis could be effectively cured and cryoglobulins also disappeared in 2 of them. But sensory polyneuropathy or Raynaud’s phenomenon did not show significant improvement during the observation period, despite the treatment with rituximab.

The most severe consequence of HCV infection and mixed cryoglobulinemia is the NHL. In one of our patients treated for DLBCL, we could see a temporary disappearance of cryoglobulins after achieving SVR. But when type II cryoglobulins reappeared, NHL also relapsed, which proved to be fatal in this case. Fortunately, in line with the current treatment guidelines, the patient with indolent marginal zone NHL could be permanently cured only with HCV eradication. During the follow-up, we did not detect cryoglobulin relapses although he also had type II cryoglobulins.

In two long-term follow-up trials, relapse or “de-novo” appearance of cryoglobulins have been observed among patients who were successfully treated with chronic HCV infection, using DAA therapy. Fayed et al. [15] could observe 12.6% vasculitis relapses in 913 patients. Importantly, half of patients with relapse required aggressive treatment, such as plasma exchange or cyclophosphamide. They concluded that relapse, even after successful DAA therapy, seems to be a big challenge. In a study from Taiwan, Chang et al. [28] determined the number of different cryoglobulin components (IgM, IgA, IgG) in their patients, but absolute amount or type of cryoglobulinemia were not reported. They similarly detected the recurrence of cryoproteins in some of their patients after obtaining cryoglobulin negative status. Surprisingly, they observed appearance of cryoglobulinemia in 9% of the originally cryoprotein negative patients, despite achieving SVR during the 4-year follow-up. It was suggested that relapse and/or “de novo” appearance could be associated either with infection or malignancy, which was also observed in our patient with DLBCL. Although, we did not reassess cryoglobulin status of our initially cryoglobulin negative patients, none of them developed any manifestations of cryoglobulinemic vasculitis during follow-up. Fluctuation of cryoprotein levels either relapse or “de novo” appearance seems to indeed suggest an independent B cell stimulation induced by the earlier HCV infection. Interestingly, only less than 2% of cryoglobulinemic patients had vasculitis in that large Asian cohort. Certainly, this is not typical for the Caucasian population. In a recent Italian publication, the PITER study by Kondili et al. [29], the authors followed a large cohort of patients with cryoglobulinemia after DAA treatment, and also observed clinical relapse in 13% of their patients after SVR. Infection as well as malignancy seemed to be two important risk factors for relapse, as was observed in our patient with aggressive NHL.

The limitation of our study is the small number of enrolled patients. However, we have interesting new details like precise typing and absolute amount of cryoproteins associated with clinical manifestations and the longest follow up period (12 years) so far after achieving SVR. We could also confirm that similar or better results can be achieved by using DAA as previously reported with IFN therapy.

In conclusion, our findings demonstrate that type II cryoglobulins are predominant in patients with mixed cryoglobulinemia and are primarily associated with clinical symptoms. After achieving SVR, patients with type III cryoglobulins show a more rapid resolution of both clinical manifestations and cryoglobulins. However, vasculitis can persist at a severity that requires immunosuppressive treatment even post-SVR. These results underscore the importance of long-term monitoring for cryoglobulinemic patients, given the potential risk of relapse of vasculitis or developing NHL, regardless of SVR status.