1
Supplement
Antibody testing:
In brief, the slides contained a biochip mosaic consisting of cryosections of primate pancreas, HEK293
cells expressing either CUZD1 or GP2, mock-transfected HEK293 control cells, and ethanol-fixed
human granulocytes[18]. Slides were embedded in PBS-buffered, DABCO containing glycerol
(approximately 10 µL per field) and examined by fluorescence microscopy with 460-490nm LED
excitation (EUROStar, Euroimmun, Lübeck, Germany). Positive and negative controls were included
throughout the analysis. A specific fluorescence at a sample dilution of 1:10 or higher was considered
positive for anti-GP2 and anti-CUZD1. Subsequently, individual sample titers were determined and
results were evaluated by two independent observers with more than ten years of professional
experience.
The use of unfixed human pancreas as well as recombinantly transformed HEK293 cells
overexpressing GP2 and CUZD1 cells enabled the simultaneous detection of antigen-specific PABs
(anti-GP2/anti-CUZD1) with a good discrimination between low positive, borderline and negative
sera. But also when tissue sections occasionally showed some interfering unspecific fluorescence due
to antibodies against cytokeratin, mitochondria, ribosomes and other antigens, discrimination
between positive and negative reactions was fostered with the additional transfected cells [18].
Sera for indirect immunofluorescence can be stored at -20°C for several months without any
significant loss of reactivity on the immunochip slides. In inflammatory bowel disease, PAB titers
appear to be very stable over the disease course since they are not influenced by different treatment
regimens [15,16].
2
Biochemical parameters of anti-GP2 IgA positive PSC patients differed significantly from anti-GP2
IgA negative patients
Anti-GP2 IgA positive Norwegian PSC patients displayed higher levels of liver transaminases and
alkaline phosphatase and APRI score (p<0.001, Suppl. Table 1), for which data were not available
from the German patient database.
Suppl. Table 1.Demonstration of PSC patients' laboratory characteristics in the Norwegian cohort
with respect to anti-GP2 IgA sero-positivity.
Characteristics of patients according to anti-GP2 IgA status
Characteristics:
Anti-GP2 IgA negative
Median (IQR):
APRI
.16 (.11-.43)
.33 (.19-.68)
<0.001
Thrombocytes [/nl]
276 (211-362)
291 (194-369)
0.371
Albumin [g/dl]
42 (40-45)
39 (33-42)
0.001
1.00 (0.94-1.17)
0.297
INR
1.04 (0.97-1.15)
Anti-GP2 IgA positive
Median (IQR):
unadjusted p-values:
Creatinine[µmol/l]
67 (58-74)
63 (57-73)
0.186
AST [U/l]
43 (33-69)
97 (67-154)
<0.001
ALT [U/l]
53 (37-124)
129 (81-194)
<0.001
Alkaline phosphatase [U/l]
158 (95-235)
321 (223-483)
<0.001
gamma GT [U/l]
212 (77-463)
273 (154-538)
0.111
Suppl. Table 1. Differences were noticed in a variety of laboratory tests (liver transaminases, albumin or
alkaline phosphatase).
[ALT=alanine aminotransferase; APRI= aspartate aminotransferase to platelet ratio index; AST=aspartate
aminotransferase; INR=international normalized ratio; IQR=interquartile range]. Significant results are
indicated in bold letters. For comparison of equality Mann-Whitney U-test was used.
3
Significant correlations between anti-GP2 IgA and bilirubin (Spearman´s r = 0.493, p<0.001) and the
MRS (Spearman´s r = 0.499, p<0.001) were detected. Regarding the correlation coefficients nearly
equal to 0.5, both correlations were of moderate intensity.
The significant correlation of anti-GP2 IgA with bilirubin and the MRS thus supported the
consideration of anti-GP2 IgA as an additional marker of disease severity.
Notably, the correlation between anti-GP2 IgA and bilirubin as well as the MRS remained significant,
when patients with biliary tract cancer were removed from the analysis (Spearman´s r (bilirubin) = 0.399,
p<0.001; Spearman´s r (MRS) = 0.404, p<0.001).
Suppl. Figure 1.Anti-GP2 IgA correlated with disease severity by means of correlation analysis with
bilirubin.
Suppl. Figure 1. Visualization of the correlation between anti-GP2 IgA titer and serum-bilirubin. Red open dots
indicate anti-GP2 IgA+ patients and blue open dots anti-GP2 IgA- ones. Anti-GP2 IgA titers are given in the
ordinate, serum-bilirubin levels in the abscissa.
Suppl. Figure 2.Anti-GP2 IgA correlated with disease severity by means of correlation analysis with
the MRS.
Suppl. Figure 2. Visualization of the correlation between anti-GP2 IgA titer and the Mayo risk score (MRS). Red
open dots indicate anti-GP2 IgA+ patients and blue open dots anti-GP2 IgA- ones. Anti-GP2 IgA titers are given in
the ordinate, the MRS is depicted in the abscissa.
Although anti-GP2 IgA titers correlated with biochemical markers of disease severity, Suppl. Figure 1
and 2 additionally demonstrate that a relevant proportion of anti-GP2 IgA positive patients did not
demonstrate excessively increased serum bilirubin or elevated Mayo risk score. Thus, a significance
of anti-GP2 IgA as an additional marker of disease severity can be derived.
4
Basal and clinical characteristics of large duct disease controls (CCA/SSC)
Table 2.CCA patients without PSC: characteristics according to anti-GP2 IgA seropositivity.
Anti-GP2 IgA negatives
Anti-GP2 IgA positives
(n=36)
(n=20)
Median (IQR):
Median (IQR):
p:
Age at time of blood samples (years)
67,08 (60,04-71,33)
64,50 (53,67-71,04)
0,281
Age at diagnosis (years)
67,04 (59,38-69,83)
63,13 (53,42-71,04)
0,417
CCA duration at serum extraction
(years)
AST [U/L]
ALT [U/L]
AP [U/L]
GGT [U/L]
Bilirubin [mg/dl]
CA 19-9 [U/mL]
CEA [µg/L]
0,50 (0,13-1,33)
27,0 (23,0-45,0)
28,5 (16,5-44,5)
159,5 (91,0-325,0)
144,0 (34,0-325,0)
0,6 (0,4-0,9)
28,9 (16,8-114,1)
1,4 (0,3-2,6)
0,08 (0,00-0,33)
86,5 (55,0-140,5)
97,5(53,5-165,5)
383,5 (346,5-888,0)
605,0(508,5-1094,0)
3,1 (0,8-5,1)
59,5 (29,7-595,7)
1,55 (0,95-5,45)
0,012
<0,001
<0,001
<0,001
<0,001
<0,001
0,147
0,637
n (%):
18 (50,0%)
n (%):
12 (60,0%)
0,472
Characteristics:
Male Gender
Localisation
 Distal bile duct
 Klatskin tumor
 intrahepatic
8 (22,2%)
20 (55,6%)
8 (22,2%)
Endoprosthesis
18 (50,0%)
[IQR=interquartile range].
1 (5,0%)
14 (70,0%)
5 (25,0%)
14 (70,0%)
0,147
5
Table 3.SSC patients’ characteristics according to anti-GP2 IgA seropositivity.
Anti-GP2 IgA negatives
Anti-GP2 IgA positives
(n=9)
(n=11)
Characteristics:
Median (IQR):
Median (IQR):
p:
Age at time of blood samples (years)
57,2 (47,2-63,0)
53,8 (39,3-59,3)
0,283
Age at diagnosis (years)
55,3 (44,6-62,0)
51,2 (39,8-58,4)
0,425
1,7 (0,7-4,1)
31,0 (26,0-36,0)
24,0 (21,0-30,0)
115,0 (88,0-193,0)
51,0 (46,0-109,0)
0,6 (0,6-0,9)
0,0 (0,0-3,5)
0,211
0,010
0,018
0,001
0,001
0,047
CCA duration at serum extraction
(years)
AST [U/L]
ALT [U/L]
AP [U/L]
GGT [U/L]
Bilirubin [mg/dl]
Male Gender
n (%):
5 (55,6%)
Etiology
 Ischemic
 Choledocholithiasis
 infectious
7 (77,8%)
0 (0,0%)
2 (22,2%)
Dominant stenosis
1 (11,1%)
[IQR=interquartile range].
66,0 (38,0-140,0)
53,0 (35,0-155,0)
616,0 (385,0-989,0)
561,0 (173,0-1551,0)
4,2 (0,7-14,3)
n (%):
11 (100,0%)
0,013
9 (81,8%)
2 (18,2%)
0 (0,0%)
2 (18,2%)
0,660
6
Low prevalence of the pancreatic autoantibody - anti-CUZD1 - in PSC
In PSC, anti-CUZD1 demonstrated a low prevalence in both the Norwegian and German PSC cohorts
(Table 1), 11.0% vs 10.6% for IgA, respectively and 10.1% vs. 4.2% for IgG, respectively, which was
similar to patients with UC, exhibiting 8.1% (IgA) and 4.8% (IgG). Anti-CUZD1 was virtually absent in
HC (Table 1). Anti-CUZD1 IgA positivity revealed no association with clinical outcomes (death/liver Tx)
in both cohorts (Suppl. Figure 3).
Suppl. Figure 3.Kaplan-Meier survival curves for PSC patients depending on anti-CUZD1 IgA status.
(A + B) Survival curves of Norwegian as well German PSC patients did not differ significantly depending on antiCUZD1 IgA status (A. Norwegian: p=0.54, Log Rank; B. German: p=0.37, Log Rank).
[CUZD1=CUB and zona pellucida-like domains 1].
Separate survival analysis for patients without a history of liver transplantation or malignancy
(CCA/GBC)
A supplementary survival analysis, which only employed “death” as a very hard and ultimate
outcome for biomarker studies analysed the anti-GP2 IgA dependent survival of patients, who did
not experienced liver transplantation or malignancy (CCA/GBC) over the disease course.
As a result we found a reproducible trend for poor survival in anti-GP2 IgA positives (see Suppl.
Figure 4) in both independent cohorts, if only death is considered in those patients without a history
of liver transplantation and without a history of biliary tract cancer.
The difference was statistically significant for survival without liver transplantation in the Norwegian
cohort (p Log Rank=0.021).
However, a clear trend for poor survival in anti-GP2 IgA positives was also seen for survival without
liver TX in the German cohort. In this regard, the missing significance for the German cohort is most
7
likely based on the reduced patient number which is included in the analysis, when the cohorts are
subdivided. In addition, one may reconsider that the use of “death” as the only outcome is a very
hard endpoint for a biomarker studies.
Suppl. Figure 4. Survival of PSC patients without liver transplantation and without malignancy
according to anti-GP2 IgA status.
(A) Survival curves of Norwegian PSC patients significantly differed, when only those patients without a history
of liver transplantation were analyzed (p Log Rank=0.021). If only those patients without a history of biliary
tract cancer were included for survival analysis, no statistical difference was detected
(p Log Rank=0.214). For all analyzes “death” was defined as the endpoint.
(B) Though not statistical significant, survival curves of German PSC patients demonstrated a remaining trend
for poor survival in anti-GP2 IgA positives. This negative trend was detectable in patients without a history of
liver transplantation (p Log Rank=0.069) and without a history of biliary tract cancer (p Log Rank=0.20).
[GP2=glycoprotein 2].
Supplementary Figures
Suppl. Figure 1.Anti-GP2 IgA correlated with disease severity by means of correlation analysis with
bilirubin.
Visualization of the correlation between anti-GP2 IgA titer and serum-bilirubin. Red open dots
indicate anti-GP2 IgA+ patients and blue open dots anti-GP2 IgA- ones. Anti-GP2 IgA titers are given
in the ordinate, serum-bilirubin levels in the abscissa.
Suppl. Figure 2.Anti-GP2 IgA correlated with disease severity by means of correlation analysis with
the MRS.
Visualization of the correlation between anti-GP2 IgA titer and the Mayo risk score (MRS). Red open
dots indicate anti-GP2 IgA+ patients and blue open dots anti-GP2 IgA- ones. Anti-GP2 IgA titers are
given in the ordinate, the MRS is depicted in the abscissa.
Suppl. Figure 3.Kaplan-Meier survival curves for PSC patients depending on anti-CUZD1 IgA status.
(A + B) Survival curves of Norwegian as well German PSC patients did not differ significantly
depending on anti-CUZD1 IgA status (A. Norwegian: p=0.54, Log Rank; B. German: p=0.37, Log Rank).
[CUZD1=CUB and zona pellucida-like domains 1].
Suppl. Figure 4. Survival of PSC patients without liver transplantation and without malignancy
according to anti-GP2 IgA status.
(A) Survival curves of Norwegian PSC patients significantly differed, when only those patients
without a history of liver transplantation were analyzed (p Log Rank=0.021). If only those patients
without a history of biliary tract cancer were included for survival analysis, no statistical difference
was detected (p Log Rank=0.214). For all analyzes “death” was defined as the endpoint.
(B) Though not statistical significant, survival curves of German PSC patients demonstrated a
remaining trend for poor survival in anti-GP2 IgA positives. This negative trend was detectable in
patients without a history of liver transplantation (p Log Rank=0.069) and without a history of biliary
tract cancer (p Log Rank=0.20).
[GP2=glycoprotein 2].