Persistence of mixed cryoglobulinemia despite cure of hepatitis C with new oral antiviral therapy including direct-acting antiviral sofosbuvir: A case series

Abstract

Objective. Obtaining a sustained virologic response (SVR) in patients with chronic hepatitis C virus (HCV) can decrease hepatic complications and be curative, however, extrahepatic manifestations including mixed cryoglobulinemia (MCN) may persist with interferon-based therapy. Our objective was to review our experience in treating patients with new oral antiviral agents and to assess common factors associated with MCN persistence despite SVR. Methods. We analyzed a case series of five patients with genotype one chronic HCV complicated by MCN who had persistence of cryoglobulins despite completion of triple therapy with oral antiviral agents (boceprivir, telaprivir or sofosbuvir). Results. Patients with cirrhosis appear to have a decreased ability to clear immune complexes. We observed that early viral response by week 8 of therapy and longer periods of undetectable virus on treatment correlated with eventual clearance of serum cryoglobulins in patients without cirrhosis. Two patients were treated with anti-B-cell agent rituximab prior to starting therapy for HCV; this did not lead to a more effective clearance of cryoglobulins. Conclusions. We suggest that a longer treatment course than the standard 24 weeks with triple therapy could aid in the clearance of these immune complexes and cryoglobulins in cirrhotics. More studies to determine the ideal duration of treatment for chronic HCV and coincident MCN are needed, especially in light of the new all oral direct-acting antiviral regimens that are now recommended for HCV treatment.

Keywords :

• Arthralgias
• chronic liver disease
• cirrhosis
• protease inhibitors
• vasculitis
• viral hepatitis

Introduction

Patients with chronic hepatitis C virus (HCV) infection are at increased risk of developing complications such as cirrhosis and hepatocellular carcinoma [1]. As a result, it is worthwhile to pursue antiviral therapy to cure HCV infection. Those patients who undergo treatment and attain a sustained virologic response (SVR) have decreased liver-related mortality, hepatocellular carcinoma and hepatic decompensation [2]. However, in addition to hepatocellular carcinoma and cirrhosis, there are multiple extrahepatic manifestations that can cause a great deal of morbidity and mortality including cryoglobulinemia, lymphoma, renal disease, porphyria cutanea tarda, lichen planus and autoimmune disorders among others [3]. These are relatively common, with up to 38% of patients having at least one extrahepatic manifestation [4,5], and have been thought to be directly related to presence of the virus. Of particular interest is the mixed cryoglobulinemia (MCN) syndrome (MCS); upwards of 50% of patients with chronic HCV have cryoglobulinemia [6], which is defined as multiple serum immunoglobulins that precipitate at temperatures ≤37°C [7-9]. MCS can cause significant morbidity for patients due to peripheral neuropathy, renal dysfunction, arthralgias/arthritis, purpura and hypocomplementemia [7,10]. It is widely accepted that curing underlying HCV is important in treating MCS [11]. Current treatment for chronic HCV genotype is now centered around all oral therapies, however, prior to the widespread availability of this regimen, the addition of a single oral antiviral agent including first-generation protease inhibitors telaprevir or boceprevir [12], or second-generation simeprevir [13-15] or direct-acting antiviral sofosbuvir [14] to pegylated interferon (PEG-IFN) and ribavirin (RBV) was widely prescribed. The safety and efficacy of this treatment regimen for HCV MCS has been evaluated in limited studies [16] and found to be effective. To date, we are unaware of any studies with the new all oral regimens investigating duration of treatment for cryoglobulinemia.

We report a case series of five patients with genotype one chronic HCV complicated by MCS who had persistence of cryoglobulins and resultant untoward clinical ramifications despite completion of standard triple therapy with the newer oral antiviral agents, including sofosbuvir.

Case 1

A 56-year-old female with a history of hypertension, restless leg syndrome and intra-abdominal surgery in 1984 due to a motor vehicle accident was originally diagnosed with genotype 1 hepatitis C (HCV) in 2004. Her initial viral load was >8.5 million IU/ml. She was followed expectantly until 2013 when she was evaluated by a neurologist for 2 years of progressive bilateral foot neuropathy. Her symptoms had persisted despite treatment with neuropathic medications including gabapentin and amitriptyline. Exam at the time revealed loss of sensation to pinprick and temperature in the lower extremities to mid-calf. While there was no evidence of large fiber neuropathy on electromyogram and nerve conduction study, a small fiber neuropathy could not be ruled out in the presence of an elevated cryoglobulin level (0.01 ppt/2 ml/48 h, normal 0.00 ppt/2 ml/48 h). One month later, she developed an erythematous, maculopapular rash with palpable purpura on her distal lower extremities concerning cryoglobulinemic vasculitic neuropathy and was started on treatment with PEG-IFN, RBV and boceprevir. She had a negative viral load by week 8 but continued to have positive cryoglobulins until 28 weeks into therapy at which time her cryoglobulin level became undetectable. Her neuropathic pain improved but did not completely resolve despite completion of treatment with SVR at 6 months and normalization of serum cryoglobulins. She continues on treatment for her neuropathic pain with gabapentin with moderate symptom control.

Case 2

A 40-year-old male with a history of depression, substance abuse and anxiety was originally diagnosed with genotype 1 HCV in 1997. His original viral load was 8.63 million IU/ml. Sixteen years after his diagnosis, the patient developed sudden onset of painful left foot drop with associated paresthesias and petechial rash in both lower extremities that became confluent in the upper thighs. His neurologic exam was notable for left lower extremity dorsiflexion and eversion weakness, sensory impairment and allodynia in a distribution consistent with common peroneal nerve involvement. Cryoglobulin levels were elevated at 0.04 ppt/2 ml/48 h and he was diagnosed with vasculitic neuropathy due to MCN associated with his chronic HCV. Induction rituximab was initiated at dose 375 mg/m². Weekly rituximab therapy was continued for 5 weeks and then the patient was started on therapy with PEG-IFN, RBV and telaprevir. After 4 weeks of anti-viral therapy, telaprevir was discontinued due to persistent viral load >1000 IU/ml. Dual therapy with PEG-IFN and RBV was continued for 3 months and then sofosbuvir was added following FDA approval and drug availability. SVR was obtained by the end of the second month of this triple therapy regimen, however, cryoglobulins were still detectable for an additional month before clearing after 15 weeks of treatment. This patient achieved SVR at 6 months and while cryoglobulins remain negative, significant neuropathic pain persisted that was somewhat refractory to medical therapy.

Case 3

A 51-year-old female with no significant past medical history was diagnosed with genotype 1a HCV incidentally in January 2013. At the initial visit, the patient reported a flat, macular rash that was distributed around the abdomen and bilateral lower extremities. There was no palpable purpura. This rash had been biopsied at an outside facility and revealed eosinophilic and suppurative folliculitis consistent with cryoglobulinemia. Urinalysis revealed hematuria and 1+ protein suggesting possible concurrent glomerulonephritis. PEG-IFN, RBV and telaprevir were started 6 months later in June 2013. Her initial viral load was 7.58 million IU/ml and this had cleared by week 4. She completed a total of 47 weeks of therapy. Despite clearing her viral load within 4 weeks, the patient had persistent cryoglobulins. Shortly after completing her antiviral therapy, the patient developed bilateral pleural effusions and fevers following breast augmentation surgery. During this hospitalization she was found to have progression of her glomerulonephritis with dysmorphic red blood cells and 3+ protein on urine dipstick. She had low complement levels and underwent a kidney biopsy which demonstrated membranoproliferative glomerulonephritis. Serum cryoglobulins were elevated at 0.19 ppt/2 ml/48 h; rheumatoid factor was elevated at 19,700 IU/ml. This was consistent with type II MCN. The patient responded well to corticosteroid therapy and was discharged on a corticosteroid taper with improving kidney function. Notably, serum protein electrophoresis was checked during this hospital stay and demonstrated a monoclonal IGM Kappa predominance. Bone marrow biopsy was consistent with monoclonal gammopathy of unknown significance but no lymphoma. Six months post-treatment, the patient obtained SVR, however, her cryoglobulins remained detectable at 0.05 ppt/2 ml/48 h. Her glomerulonephritis has remained in remission following discontinuation of corticosteroids but her cryoglobulins have not become undetectable.

Case 4

A 49-year-old female with medical history significant for HCV cirrhosis, cryoglobulinemia with complications including vasculitic rash with palpable purpura and chronic kidney disease stage III, hypertension, hyperlipidemia and coronary artery disease was hospitalized in May 2013 for non-ST elevation myocardial infarction (NSTEMI) requiring placement of a drug-eluting stent. This was complicated by development of dyspnea and acute kidney injury. Her creatinine had increased from 1.8 to 2.5 mg/dl, rheumatoid factor was elevated at 3600 IU/ml, cryoglobulins 0.08 ppt/2 ml/48 h, complement levels were low, HCV viral load elevated at 15.7 million (IU/ml) and urinalysis showed an active sediment and proteinuria. Renal biopsy was deferred due to bleeding risk; regardless, the patient was diagnosed with likely membranoproliferative glomerulonephritis and began treatment with weekly rituximab at dose 375 mg/m² for four doses. Renal function returned to baseline after therapy with rituximab and corticosteroids. Several weeks after discharge, she started therapy for HCV with PEG-IFN. RBV was added to regimen in July 2013 after her significant anemia improved. She was maintained on dual therapy until January of 2014 when sofosbuvir was added due to persistent viral load (<43 IU/ml) and cryoglobulinemia (0.05 ppt/2 ml/48 h). After 2 weeks of triple therapy, she cleared her viral load and then completed 12 weeks of triple therapy with sofosbuvir without clearing her cryoglobulinemia. Renal function has remained stable with a creatinine of 1.3 mg/dl and an inactive urine sediment at the time SVR was obtained. The patient remains off treatment for cryoglobulinemia at that time having been tapered off corticosteroids.

A 65-year-old female was diagnosed with genotype 1a HCV in 2000. At that time, she was treated with PEG-IFN and RBV for 1 year with clearance of virus before relapsing. In 2010, she developed periorbital and pedal edema in the setting of RBC casts, normocytic anemia and positive cryoglobulinemia. This was thought to be consistent with chronic HCV-related MCN and a renal biopsy demonstrated membranoproliferative glomerulonephritis. During the work-up of her anemia, she was also diagnosed with a low-grade lymphoma, which was subsequently treated with weekly rituximab infusions at dose 375 mg/m² for 4 weeks. In December 2012, she again had a flare of symptoms with worsening anemia, fatigue, edema and ankle rash. She received rituximab again for two doses and her HCV viral load increased from 3.46 million IU/ml in December 2011 to 60 million IU/ml in February 2013. She subsequently underwent treatment with PEG-IFN, RBV and sofosbuvir in January 2014. By week 8, she had cleared her virus and by week 12 she had cleared her cryoglobulins. SVR was obtained 6 months post-treatment for her HCV. The patient has restarted lymphoma treatment with maintenance dosing of rituximab every 6 months. Her cryoglobulins have remained negative.

Table 1 shows a summary of the salient features of each of the above five cases.

Table 1. Summary of patient demographics and outcomes.

	Therapy	Pretreatment VL	Initial cryoglobulin	Cryomanifestations	Time to first ND VL	Time to clear cryoglobulins	Treatment duration	SVR
Patient 1	PEG-IFN, RBV, boceprevir	8480000	0.01	Neuropathy	8 weeks	Did not clear (55 weeks?)	28 weeks	Yes
Patient 2	PEG-IFN, RBV, telaprevir	8630000	0.09	Vasculitis	Did not clear	Did not clear
	PEG-IFN, RBV	1410	0.01	Vasculitis	Did not clear	Did not clear
	PEG-IFN, RBV, sofosbuvir	2070	0.01	Vasculitis	6 weeks	12 weeks	15 weeks	Yes
Patient 3	PEG-IFN, RBV, telaprevir	7580000	0.07	MPGN, monoclonal spike	4 weeks	Did not clear	47 weeks	Yes
Patient 4	PEG-IFN	13100000	0.19	MPGN	Did not clear	Did not clear
	PEG-IFN, RBV	2334	0.1	MPGN	Did not clear	Did not clear
	PEG-IFN, RBV, sofosbuvir	<43	0.05	MPGN	2 weeks	Did not clear	12 weeks	Yes
Patient 5	PEG-IFN, RBV, sofosbuvir	8690000	0.03	MPGN, low grade lymphoma?	8 weeks	12 weeks	12 weeks	Yes

Abbreviations: HCV = Hepatitis C virus; MPGN = Membranoproliferataive glomerulonephritis; ND = Non detectable; PEG-IFN = Pegylated interferon; RBV = Ribavirin; SVR = Sustained virologic response; VL = Viral load.

Discussion

We report five patients with genotype 1 HCV complicated by MCS that despite completion of at that time standard triple therapy with oral antiviral agents, PEG-IFN and RBV had persistence of cryoglobulins. This manifested as clinically significant vasculitis, renal dysfunction from membranoproliferative glomerulonephropathy, neuropathy and lymphoma. The unwanted clinical effects of persistent cryoglobulinemia raises questions about how best to treat those with HCV-associated MCS, especially in light of the multitude of new oral therapies.

MCS is most commonly found in association with HCV, HBV, HIV or other chronic liver disease [6,17,18]. Of particular interest is chronic HCV because up to 80% of patients with MCS can have positive HCV antibodies [8]. The pathophysiology of MCS is secondary to antigen–antibody complexes depositing in small vessels leading to palpable purpura, renal disease, arthralgias, neuropathy and other manifestations [19]. In chronic HCV, this immune complex consists of HCV, anti-HCV IgG and rheumatoid factor. This can be driven by B-lymphocyte expansion which has been shown to be responsible for both autoantibodies and immune complex production [7,19] provoked by the HCV virion itself, which can be found in high concentrations in the cryoprecipitate [6]. Some have gone so far as to describe a ‘HCV syndrome’ in which HCV-induced B lymphocyte proliferation leads to the development of multiple autoimmune lymphoproliferative disorders across multiple organ systems [20]. Prolonged liver disease and cirrhosis can lead to an impaired ability to clear these immune complexes, thus increased levels of cryoglobulins, and therefore more tissue and small vessel deposition [6]. In fact, cryoglobulins have been shown to be an important prognostic indicator using regression modeling for the risk of cirrhosis secondary to chronic HCV as they are highly significant associated with cirrhosis [21].

Treatment of chronic HCV is important to avoid complications such as cirrhosis, hepatocellular carcinoma and decompensated liver failure [1]. If the presence of HCV is responsible for inciting the production of cryoglobulins, then it would seem reasonable to proceed with treatment for HCV when patients are experiencing adverse effects secondary to MCS. Our five patients in this series eventually underwent treatment for this reason. In this subset of patients, the inclusion or addition of oral, bioavailable protease inhibitors or polymerase inhibitor sofosbuvir substantially enhanced rates of virological response when it was combined with PEG-IFN and RBV. The goal of treating HCV has typically been to attain a SVR by eradicating HCV RNA, but should we also focus on clearing cryoglobulins in those affected by this disease?

In our series of patients, we found that cryoglobulin clearance seems to lag behind viral load clearance. While there is no clear mechanism to explain this finding, we propose that patients with more advanced chronic HCV (i.e., stage 3 or 4 fibrosis or cirrhosis) have a decreased ability to clear immune complexes and thus a longer treatment course than the currently recommended duration of therapy could aid in the clearance of these immune complexes and cryoglobulins Table 2. This is demonstrated by patient 3 who had stage 4 fibrosis and rapid viral response by week 4 but never cleared her cryoglobulins. Furthermore, time to viral clearance may predict which patients eventually obtain an undetectable level of cryoglobulins and ideally resolution of the clinical symptoms of MCN in the absence of advanced liver disease. All patients in our series who had a negative HCV viral load by 8 weeks of therapy and no evidence of stage 4 fibrosis/cirrhosis eventually cleared their cryoglobulins. Interestingly, clearance of cryoglobulins did not necessarily ensure resolution of clinical symptoms.

Table 2. Biopsy findings pre treatment.

	Fibrosis stage	Grade of necroinflammatory changes
Patient 1^a	-	-
Patient 2	3	1
Patient 3	4	3
Patient 4	3	3
Patient 5	3	2

^aPatient 1 was without liver biopsy.

Of additional interest are patients 4 and 5 who both underwent therapy with the anti-CD20 drug rituximab before beginning triple therapy for membranoproliferative glomerulonephritis and low-grade lymphoma, respectively. Patients who have severe MCS manifested as glomerulopnephritis and vasculitis often require therapy with rituximab [11,22]. Rituximab blocks rheumatoid factor and immune complex production by acting on proliferating B cells [19,23]. Interestingly in these cases, treatment with rituximab before undergoing triple therapy did not prevent cryoglobulins from returning and did not provide a more rapid clearance of cryoglobulins. And while there is inherent selection bias in this observation as rituximab is typically reserved for the most aggressive forms of MCN, this finding does support that of Benstead et al. [24]. who recently questioned the utility of rituximab therapy in a recent Cochrane review which cited insufficient data to make evidence-based treatment decisions for extrahepatic manifestations of cryoglobulinemia and HCV.

One limitation of our study is the relative heterozygosity of the five patients, that is, the treatment regiments were all essentially different in that some contained first-generation protease inhibits, others, polymerase inhibitors. Two patients were treated with rituximab. However, this just demonstrates the need for individualized therapy for these difficult-to-manage patients. In fact, Ferri et al. [19] confirm this by validating that combined antiviral and immunosuppressive therapy should be tailored for each individual patient according to the severity of clinical symptoms. This heterozygosity may make it difficult to draw concrete conclusions, however, what is certain is that regardless of the regimen, attainment of HCV RNA negativity does not always confer resolution of cryoglobulinemia.

Conclusion

Data regarding efficacy of triple antiviral therapy in managing HCV MCS patients are limited and to our knowledge no studies have investigated the role of the new all oral direct-acting antiviral regimens for patients specifically with MCN. Our patient series suggests that while the addition of orally, bioavailable protease inhibitors to PEG-IFN and RBV results in a more substantial virologic response, attainment of HCV RNA negativity does not always confer resolution of cryoglobulinemia. We believe that opportunities exist specifically in the HCV MCS population to further study alternative treatment regimens/durations in an attempt to improve the clinical ramifications of MCS. With newer non-IFN-based therapies available now, this may reduce the resultant antibody production in HCV and during treatment. Future studies may be necessary to see if this will affect the rate of cryoglobulin clearance and the utility of using serum cryoglobulins as a prognosticator for which patients may be at higher risk for cirrhosis and the resultant complications. As more regimens are striving for shorter durations of therapies on the order of several months, caution should be taken in those patients with MCS, as they may still require longer regimens to clear the extrahepatic manifestations of HCV infection.

Declaration of interest

This work was supported in part by a grant from the National Institutes of Health. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.