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Serum IgG4 cut-off of 70 mg/dL is associated with a shorter time to cirrhosis decompensation and liver transplantation in primary sclerosing cholangitis patients

Abstract BACKGROUND Primary sclerosing cholangitis (PSC) is an immune-mediated biliary disorder of unknown etiology with no effective treatment. The purpose of this study was to better prognosticate the development of cirrhosis, decompensation, and requirement for liver transplantation (LT) in PSC patients based on serum immunoglobulin G4 (IgG4) levels. METHODS A retrospective chart review was conducted on PSC patients seen at the University of Alberta Hospital between 2002 and 2017. PSC patients were categorized as high IgG4 group (≥70 mg/dL) or normal IgG4 group (<70 mg/dL). Laboratory parameters, clinical characteristics, and outcomes were compared between the groups. RESULTS One hundred and ten patients were followed over a mean period of 7.3 (SD 5) years. Seventy-two patients (66%) were male, the mean age at diagnosis of PSC was 35 (SD 15) years, and inflammatory bowel disease (IBD) was present in 80 patients (73%). High IgG4 levels were found in 37 patients (34%). PSC patients with high IgG4 had a shorter mean cholangitis-free survival time (5.3 versus 10.4 years, p = 0.02), cirrhosis-free survival time (8.7 versus 13.0 years, p = 0.02), and LT-free survival time (9.3 years versus 18.9 years, p <0.001). IgG4 ≥70 mg/dL was independently associated with liver decompensation and LT-free outcomes. A cut-off IgG4 value of ≥70 mg/dL performed better than a cut-off value of ≥140 mg/dL to predict time to LT (area under the curve [AUC] 0.68, p = 0.03, sensitivity 72%, specificity 78%). CONCLUSIONS Serum IgG4 ≥70 mg/dL in PSC predicts a shorter time to cirrhosis decompensation and LT.


INTRODUCTION
Primary sclerosing cholangitis (PSC) is a progressive chronic liver disease that leads to destruction of the biliary tract, chronic cholestasis, multifocal biliary strictures, and cirrhosis (1). The incidence rate of PSC is 0.77 per 100,000 person-years, and up to 70% of PSC patients suffer from concomitant inflammatory bowel disease (IBD) (2,3). In the absence of effective therapy, over 50% of patients require liver transplantation (LT) within 10-15 years of symptom onset (4). Furthermore, 20%-25% of PSC patients will exhibit recurrence 10 years post-LT (5).
As the disease course is variable and most PSC patients will progress to LT, several prognostic models for predicting clinical outcomes in PSC have been developed. The revised Mayo risk score is the most commonly used model; however, it has inherent limitations (1,6). Other models incorporating non-invasive fibrosis biomarkers and magnetic resonance imaging (MRI)/elastography also exist (7)(8)(9). In 2006, Mendes et al demonstrated that PSC patients with an elevated serum IgG4 had higher bilirubin, alkaline phosphatase, PSC Mayo risk scores, and a shorter time to LT (1.7 versus 6.5 years, p = 0.0009) (10).
PSC patients with elevated serum immunoglobulin G4 (IgG4) levels likely represent a PSC subtype, which can be identified in 9%-22% of PSC patients depending on the cut-off level used to demarcate an elevated IgG4 (11). While some population-based studies have supported the use of serum IgG4 as a prognostic marker for liver outcomes in PSC patients, others have not (11)(12)(13)(14). Due to the inconsistency and relative lack of reports, we aimed to better define the relevance of assessing IgG4 levels in PSC patients. Specifically, the primary purpose of this study was to prognosticate the development of complications related to portal hypertension and the requirement for LT in PSC patients based on the hypothesis that PSC patients with elevated serum IgG4 levels have worse outcomes and more aggressive disease.

Study patients
A retrospective chart review was conducted on PSC patients followed at the University of Alberta between 2002 and 2017. The University of Alberta's human research ethics board approved this study in January 2017 (Pro00069785). PSC diagnosis was based on standard criteria including (1) cholestatic liver enzyme elevation, (2) characteristic magnetic resonance cholangiopancreatography or endoscopic retrograde cholangiopancreatography findings, and (3) exclusion of secondary causes of cholangitis (15). A diagnosis of PSC was confirmed on chart review if no other typical features of IgG4related disease were present. PSC patients who had at least one serum IgG4 level prior to LT or last follow-up were included in the study. In patients with multiple IgG4 levels, the maximum IgG4 level was chosen. Serum anti-smooth muscle antibody (positive titre defined by at least 1:20), IgG, IgG4, aspartate aminotransferase (AST), aspartate aminotransferase (ALT), alkaline phosphatase (ALP), bilirubin, international normalized ratio (INR), albumin, creatinine, platelet count, and concomitant IBD were recorded. Moreover, PSC phenotype (intrahepatic/extrahepatic involvement), first episode of cholangitis (defined as abdominal pain, fever, jaundice, and a dilated common bile duct on imaging), initial requirement for biliary endoscopic treatment, and development of cholangiocarcinoma (CCA), as well as the presence of cirrhosis, liver decompensation, and the requirement for LT, were also documented.
The presence of cirrhosis was defined by histological cirrhosis in liver tissue examination with demonstration of a complete regenerative nodule (16), an increased FibroScan reading (>14 kPa) (FibroScan, Echosens, Paris, France), radiological findings of cirrhosis by computed tomography (CT) or MRI, and/or the development of portal hypertension or its complications. Decompensation date was recorded as the first episode of variceal bleeding, ascites, or hepatic encephalopathy. Model for end-stage liver disease (MELD) scores and PSC Mayo risk scores were calculated at the time of max IgG4 ±1 month (ie, within either 4 weeks prior to the time when max IgG4 level was drawn or within 4 weeks after it was drawn). The PSC Mayo risk score was calculated as per http:// www.mayoclinic.org/gi-rst/mayomodel3.html. Patients with autoimmune pancreatitis (AIP), IgG4 associated cholangitis (IAC), primary biliary cholangitis (PBC), or overlap syndrome with autoimmune hepatitis were excluded (17).

IgG4 determination
IgG4 levels at the University of Alberta were quantified by the nephelometric method using Binding

Patient characteristics
During this period, we evaluated 445 PSC patients seen in our autoimmune liver disease clinic. A total of 325 of the 445 patients were excluded as they did not have at least one IgG4 test. After chart review, 10 more patients were excluded as they had diagnoses of autoimmune pancreatitis (1), PBC (1), IgG4 associated cholangitis (3), and autoimmune overlap (5) (Figure 1.) Since a significant number of patients were not eligible for inclusion, we briefly analyzed patients included (n = 110) and excluded (n = 335) from this study. We did not find significant differences in relation to age at PSC diagnosis (mean 34. 9  between these two groups. One hundred and ten PSC patients remained, and 34% were classified as having a high IgG4. All patients were followed over a period of 7.3 (SD 5 years) and 73% had concomitant IBD (Table 1).

PSC phenotype
The presence of dominant strictures, the location of biliary involvement, the prevalence of cholangitis and the need for endoscopic intervention did not differ between PSC patients with high IgG4 and PSC patients with normal IgG4 (Table 1).
Site reagent (The Binding Site Group Ltd., Birmingham, UK). The "normal" IgG4 level range varied depending on whether the assay was conducted at the University of Alberta hospital (7-74 mg/ dL) or community hospitals in Alberta (4-164 mg/ dL, 3-200 mg/dL, <139 mg/dL). The cut-off for an elevated IgG4 was determined to be ≥70 mg/dL based on a previously published abstract where a serum IgG4 level ≥70 mg/dL had the best operating characteristics for predicting time to LT (area under the curve [AUC] 0.76, p = 0.04) in 46 PSC patients, who are also included in this cohort (18).

Statistical analysis
Data are reported as mean and standard deviation (SD) for continuous variables and as a percentage for categorical variables. Time to cholangitis, biliary endoscopic treatment, CCA, portal hypertensive complications, cirrhosis, and LT-free survival were calculated by Kaplan-Meier methods and compared by the log-rank (Mantel-Cox) test. Significant predictors of the outcomes were determined using univariate and multivariate Cox proportional hazard models, and the results were reported as hazard ratios (HR) with 95% CIs. A receiver operating characteristics (ROC) curve was plotted to measure how serum IgG4 performed in predicting LT within 10 years. Model capability to distinguish between outcome groups was assessed using the AUC. The value with the highest Youden index (sensitivity + specificity − 1) was considered as the optimal cut-off.   Figure 2).

Cholangitis, endoscopic treatment, and cholangiocarcinoma
The mean cholangitis-free survival time in patients with high serum IgG4 was 5.3 years (95% CI 3.9-10.8 years) compared to 10.4 years (95% CI 9.1-11.8 years) in those with normal IgG4 levels (Figure 3a, p = 0.02). On univariate analysis, a higher risk of cholangitis was associated with high serum IgG4 (HR 2.68, 95% CI 1.16-6.22, p = 0.02), Last, the mean LT-free survival in patients with elevated IgG4 was 9.3 years (95% CI 8.0-10.7) higher INR, higher IgG, lower albumin, and higher MELD scores. Furthermore, a serum IgG4 level ≥70 mg/dL predicted a shorter time to cholangitis, cirrhosis, and LT as well as was independently associated with cirrhosis decompensation and LT outcomes. Contrary to serum IgG4 cut-offs published previously in the literature (Table 3), a serum IgG4 ≥70 mg/dL better predicted a shorter time to LT within 10 years from the time of PSC diagnosis compared to a serum IgG4 ≥140 mg/dL.
Our study confirms that there is a subset of PSC patients with elevated serum IgG4 who do not meet the criteria for IAC and may have a worse prognosis. As in Mendes et al (10)

DISCUSSION
In our cohort of PSC patients, we identified that patients with a serum IgG4 ≥70 mg/dL had a younger age at diagnosis, higher alkaline phosphatase,   (20). They demonstrated that PSC patients with increased IgG4 positive plasma cells were more likely to display recurrence of disease post-transplant and had a significantly shorter time to transplant. In contrast, although nearly half (58/122) of PSC liver explants from a Canadian cohort had evidence of hilar IgG4-positive staining, PSC patients with increased IgG4 staining were not predisposed to increased recurrence post-transplantation (21). The potential role of IgG4 in PSC, whether it be in serum or tissue, is still debatable. IgG4 may play a pathogenic role, such as in pemphigus vulgaris, or reflect an epiphenomenon of an inflammatory response (22). Interestingly, studies have also demonstrated that IAC and PSC have contrasting host-microbe interactions that may be involved in disease pathogenesis (23). The higher levels of IgG4 may possibly reflect changes in the bile microbiota and subclinical cholangitis in patients with more advanced liver disease.
One of the strengths of our study was that we had a well-phenotyped cohort of PSC patients followed for almost a decade. Although there is a possibility that IgG4-associated cholangitis (IAC) patients were misclassified as PSC patients with elevated IgG4 in our population, it is reassuring that the mean age of diagnosis of our patients was 35 years, with 73% of patients having concomitant IBD and less than 5% of patients having pancreatic involvement, which is more reflective of PSC patients rather than IAC patients (19,24). Furthermore, we are the first group to observe the In summary, we have demonstrated that a serum IgG4 ≥70 mg/dL in PSC patients is a non-invasive prognostication marker that predicts worse liver outcomes and, more specifically, a shorter time to cirrhosis decompensation and LT. Moreover, our findings suggest that the definition of what constitutes an elevated IgG4 in PSC patients should not be directly extrapolated from criteria developed for IgG4-related disease. It is evident that an elevated IgG4 >140 mg/dL used for diagnostic purposes in IAC may not be the best predictor of poor prognosis in PSC patients. As this is a single-centre cohort study, these results will need to be replicated in other centres and ideally in a prospective multicentre trial. While the significance of IgG4 remains speculative, we propose that serum IgG4 be used as risk stratification in therapeutic trials to potentially help PSC patients with a more aggressive phenotype. behaviour of serum IgG4 levels longitudinally. We have demonstrated that though serum IgG4 levels fluctuate, most PSC patients remained in either the high IgG4 group or normal IgG4 group over time. Last, a serum IgG4 level of >140 mg/dL is an arbitrary cut-off to define elevated IgG4 in PSC and has been adopted from the HISORt criteria for IgG4-related disease (25). We demonstrated better operating characteristics using a cut-off of ≥70 mg/dL to predict liver outcomes compared to 140 mg/dL.
Nevertheless, there are also several limitations to our study. Data was collected retrospectively and is subject to tertiary care referral centre bias. Therefore, our results may not apply to the general PSC population. Furthermore, neither the number nor the timing of IgG4 determinations was standardized. Reassuringly, the time from PSC diagnosis to maximum IgG4 level did not differ between the high and normal IgG4 groups (Table 1). Data from Bjornsson et al revealed that prednisone treatment in PSC patients with elevated IgG4 can result in decreasing bilirubin, alkaline phosphatase and IgG4 levels (26). Unfortunately, it is unclear what type of immunosuppression, if any, was being used at the time IgG4 levels were drawn in our cohort. It is also possible that episodes of cholangitis result in elevations of serum IgG4, and therefore our data demonstrating a shorter cholangitis-free survival time in patients with IgG4 ≥70 mg/dL needs to be interpreted with caution. Moreover, the use of ursodeoxycholic acid and dosing was not reliably captured on chart review. These could have affected the endpoints of cirrhosis and LT, as highdose UDCA (28-30 mg/kg) has been associated with worse clinical outcomes (27). Finally, we had a relatively small population of patients in our cohort compared to other studies published with 200-300 PSC patients, and only a select number of PSC patients had IgG4 values drawn. Although selection bias is present, reassuringly, there were no differences with respect to gender, age at PSC diagnosis, and liver transplant-free survival between our entire PSC cohort followed at the University of Alberta and the subset population who had IgG4 levels drawn. Unfortunately, we could not analyze the performance of new scores to predict clinical outcomes in PCS, such as the PREsTo score (28), because the available calculator only provided the percent risk of hepatic decompensation and not the raw number for the score.