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A Changing Natural History of Primary Biliary Cholangitis and Its Influence on Risk Stratification
by
Carla Fiorella Murillo Perez
A thesis submitted in conformity with the requirements for the degree of Master of Science
Institute of Medical Science University of Toronto
A changing natural history of primary biliary cholangitis and its
impact on risk stratification
Carla Fiorella Murillo Perez
Master of Science
Institute of Medical Science University of Toronto
2018
Abstract
We sought to describe temporal trends in the presenting characteristics and clinical course of
primary biliary cholangitis (PBC) from the 1970s to 2014 in a large international cohort of
patients. There was a 10-year increase in age at diagnosis and the proportion of patients
presenting with a mild biochemical and histological disease stage increased. Furthermore, recent
decades had improved decompensation and transplant-free survival rates. Since most patients
presented with normal bilirubin, we aimed to evaluate whether normal bilirubin is associated
with transplant-free survival. In patients with normal bilirubin at 1 year, the threshold with the
highest ability to predict liver transplantation or death was 0.6×ULN. Patients with normal
bilirubin, yet above this threshold, had a 2.1-fold increase in risk. In conclusion, the natural
history of PBC has changed over time and bilirubin levels ≤0.6×ULN are associated with the
lowest risk for liver transplantation or death in patients with PBC.
iii
Acknowledgments I would like to thank my family and friends for supporting and helping me on this journey
towards the completion of my Master’s.
I would like to express my gratitude towards my supervisor and mentors for their guidance,
advice, and support: Harry Janssen, Bettina Hansen, Jordan Feld, and Aliya Gulamhusein. It has
truly been a pleasure working with all of you.
I would also like to thank my international co-workers: Jorn Goet, Willem Lammers, Henk van
Buuren, Maren Harms, and Adriaan van de Meer who have collaborated with me.
I would also like to also acknowledge the MSc/PhD students at the Toronto Centre for Liver
Disease who have supported me: Seng Liem, Lisette Krassenburg, Mina Farag, Hooman
Zangneh, Surain Roberts, Jason Lau, and Hannah Choi.
I would like to thank the GLOBAL PBC Study Group members for welcoming me as a new
member and supporting my research.
iv
Table of Contents Acknowledgments.......................................................................................................................... iii
Table of Contents ........................................................................................................................... iv
List of Tables ............................................................................................................................... viii
List of Figures ..................................................................................................................................x
List of Abbreviations .................................................................................................................... xii
Chapter 1 : Literature Review ..........................................................................................................1
2.2.3 Rationale for hypothesis ........................................................................................34
vi
Chapter 3 : Study 1 ........................................................................................................................36
Milder Disease Stage in Patients with Primary Biliary Bholangitis Over a 44-year Period: A Changing Natural History ..........................................................................................................36
3.5 Supplementary Tables and Figures ....................................................................................55
Chapter 4 : Study 2 ........................................................................................................................68
Bilirubin is Predictive of Transplant-free Survival Even Within the Normal Range in Patients with Primary Biliary Cholangitis .....................................................................................68
4.3.1 Study population characteristics ............................................................................72
vii
4.3.2 Normal bilirubin quartiles are associated with liver transplant-free survival ........74
4.3.3 Bilirubin threshold within the normal range ..........................................................75
4.3.4 The risk for liver transplantation or death increases at bilirubin levels of 0.6×ULN ................................................................................................................79
4.3.5 Patients who remain below 0.6×ULN over time have good long-term prognosis ................................................................................................................80
4.3.6 The proportion of patients with bilirubin ≤0.6×ULN increased over time ............80
List of Tables Table 1-1. Various response criteria to UDCA for PBC .............................................................. 25
Table 3-1. Demographic and clinical characteristics of PBC patients at study entry over calendar
time ............................................................................................................................................... 41
Table 3-2. Multivariable logistic regression for the attainment of biochemical response
according to Paris-Ia (n=2283) ...................................................................................................... 48
Table S3-1. Distribution of PBC patients across calendar time and center ................................. 55
Table S3-2. Calendar time trends in patients with a maximum lag of 2 years between diagnosis
and study entry .............................................................................................................................. 57
Table S3-3. Factorial ANOVA analysis of age at diagnosis over calendar time adjusting for sex
Table 4-1. Characteristics of PBC patients in each normal bilirubin cohort ................................ 73
Table 4-2. Multivariable Cox regression analyses of various bilirubin thresholds in patients with
normal bilirubin at 1 year to evaluate performance for the prediction of liver transplantation and
death .............................................................................................................................................. 76
ix
Table S4-1. Multivariable Cox regression analyses of various bilirubin thresholds in patients
with normal bilirubin at time zero to evaluate performance for the prediction of liver
transplantation and death .............................................................................................................. 85
Table S4-2. Multivariable analysis of 0.6×ULN threshold at 1 year in various sub-groups ....... 87
x
List of Figures Figure 3-1. Age at diagnosis of PBC patients across different decades. ..................................... 43
Figure 3-2. Study entry characteristics associated with disease severity of patients diagnosed in
different decades.. ......................................................................................................................... 45
Figure 3-3. Response rates to ursodeoxycholic acid (UDCA) therapy over calendar time. ........ 47
Figure 3-4. Time-to-event analyses of decompensation, hepatocellular carcinoma (HCC), and
liver transplantation or death over calendar time. ......................................................................... 50
Figure S3-1. Mean age at diagnosis over calendar time stratified by A) Center (each line
corresponds to an individual center); B) Sex; and C) Biochemical disease stage. ....................... 60
Figure S3-2. Absolute number of patients according to age at diagnosis and over calendar time.
Data represented as mean (standard deviation), n (%), or median (interquartile range). Primary biliary cholangitis, PBC; AMA, antimitochondrial antibody; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ULN, upper limit of normal; ALT, alanine aminotransferase; LLN, lower limit of normal; APRI, AST to platelet ratio index; UDCA, ursodeoxycholic acid. aAge at diagnosis not available for one patient in 2000-2009 cohort. bAMA status was available for 4686 (97.5%) patients. cALP, bilirubin, AST, and ALT were log transformed prior to analyses and availability for laboratory values is as follows: ALP: 3560 (74.1%); Bilirubin: 3595 (74.8%); AST: 3460 (72.0%); ALT: 3007 (62.6%); Albumin: 3039 (63.2%); Platelet count: 2769 (57.6%) dThe cut-point APRI >0.54 at baseline is predictive of liver transplantation or death (Trivedi et al., 2014) eBiochemical disease stage classification according to Rotterdam criteria (ter Borg et al., 2006) was available in 2958 (61.6%) patients. fHistological disease stage at diagnosis according to Ludwig et al. and Scheuer (Ludwig et al., 1978; Scheuer, 1967) classification was available in 2217 (46.1%) patients. gUDCA therapy status was available for 4727 patients (98.4%).
3.3.2 Age and sex trends
The mean age at diagnosis increased incrementally from 46.9 ± 10.1 years in the 1970s to 57.0 ±
12.1 years from 2010 onward (P < 0.001, Figure 3-1A). This trend was consistent across center,
sex, and biochemical disease stage (Figure S3-1A-C). The effect of calendar time on the
increase in age at diagnosis remained significant (P < 0.001) after correcting for sex (Table S3-
3). Furthermore, the age distribution of patients notably changed over the investigated decades (P
< 0.001, Figure 3-1B). The proportion of patients aged 50-59 years at diagnosis remained
relatively stable across the years, whereas the proportion of patients <50 years of age decreased
and patients ≥60 years of age increased. There was no significant temporal trend in the female to
male ratio, which remained approximately 9:1 (Table 3-1).
Figure 3-1. Age at diagnosis of PBC patients across different decades. A) Mean age (± standard
deviation) at diagnosis (dots) and estimated marginal means (squares) obtained after adjusting
for sex. B) The distribution of age groups over calendar time.
Year of diagnosis
Age
at d
iagn
osis
(yea
rs)
1970-1979 1980-1989 1990-1999 2000-2009 ≥ 201020
30
40
50
60
70
Raw dataEstimated marginal means
P < 0.001
Perc
enta
ge o
f pat
ient
s
1970-19791980-19891990-19992000-2009 ≥ 20100
20
40
60
80
100
<3030-3940-4950-5960-69≥70
Year of diagnosis
Age at diagnosis (years)
A) B)
43
44
3.3.3 Liver biochemistry and serological status
The proportion of patients that were AMA-positive did not significantly differ across the
investigated decades (Table 3-1). Median alkaline phosphatase and bilirubin values (× upper
limit of normal) at study entry decreased, while circulating platelet counts were noted to increase
(P < 0.001), which collectively suggests a less advanced disease stage. The proportion of patients
with alkaline phosphatase values below 2 × the upper limit of normal increased gradually from
30.0% in the 1970s to 63.1% from 2010 onward (P < 0.001) (Figure 3-2A). The proportion of
patients with normal serum bilirubin concentrations also increased from 51.1% in the 1970s to
77.6% in the 1990s, after which it remained relatively stable (P < 0.001) (Figure 3-2B).
Furthermore, a reduced percentage of patients with aspartate aminotransferase/platelet ratio
index >0.54 at study entry was observed (Table 3-1).
45
Figure 3-2. Study entry characteristics associated with disease severity of patients diagnosed in
different decades. A) Percentage of patients with alkaline phosphatase (ALP) above or below 2
times the upper limit of normal (×ULN). B) Percentage of patients with bilirubin above or below
1×ULN. C) Percentage of patients corresponding to each biochemical stage according to
Rotterdam criteria (ter Borg et al., 2006); mild (normal albumin and bilirubin), moderate
(abnormal albumin or bilirubin), advanced (abnormal albumin and bilirubin). D) Percentage of
patients corresponding to each histological stage at diagnosis according to Ludwig et al.’s (1978)
and Scheuer’s (1967) classification: mild (stage I and II) or advanced (stage III and IV).
The proportion of UDCA-responders according to Paris-I, Toronto, Paris-II, Rotterdam, and
GLOBE score criteria increased over the investigated decades (P < 0.001), but not according to
Barcelona criteria (Figure 3-3, Table S3-4). Importantly, this trend remained true in patients
who did not meet the individual criteria at baseline (Table S3-5). In a multivariable logistic
regression, calendar time was not a significant predictor for UDCA-response according to Paris-I
criteria (Table 3-2). Response was associated with an increased age at diagnosis, and lower
alkaline phosphatase and bilirubin levels (P < 0.001). Additionally, calendar time was also not a
47
significant predictor for UDCA-response according to Toronto, Paris-II, Rotterdam, and GLOBE
score criteria.
Figure 3-3. Response rates to ursodeoxycholic acid (UDCA) therapy over calendar time.
Response was determined according to various published criteria: Barcelona, Paris-I, Rotterdam,
Toronto, Paris-II, and the GLOBE score (Corpechot et al., 2008, 2011; E. M. Kuiper et al., 2009;
Kumagi et al., 2010; Lammers et al., 2015; Parés et al., 2006). Response rates according to all
criteria were significantly different over calendar time (P < 0.001), except Barcelona criteria (P =
0.19).
Year of diagnosis
Perc
enta
ge o
f pat
ient
s
1970-19791980-19891990-19992000-2009 ≥20100
20
40
60
80
100
Paris-I
Paris-II
Rotterdam
Barcelona
GLOBE score
Toronto
Table 3-2. Multivariable logistic regression for the attainment of biochemical response
according to Paris-Ia (n=2283)
Variable OR 95% CI P-value
Male sex 0.90 0.63-1.29 0.58
Year of diagnosis 0.67
1970-1979 1.00
1980-1989 0.80 0.37-1.71 0.66
1990-1999 1.01 0.44-2.37 0.96
2000-2009 0.97 0.40-2.32 0.94
≥2010 0.92 0.33-2.57 0.88
Age at diagnosis 0.04
<30 1.00
30-39 1.29 0.53-3.15 0.57
40-49 1.41 0.60-3.33 0.44
50-59 1.95 0.82-4.59 0.13
60-69 2.06 0.86-4.96 0.11
≥70 2.06 0.82-5.21 0.13
Log bilirubin (×ULN) 0.01 0.01-0.02 <0.001
Log ALP (×ULN) 0.12 0.08-0.18 <0.001
Difference between diagnosis and study entry (years) 0.98 0.94-1.03 0.44
OR, odds ratio; CI, confidence interval; ULN, upper limit of normal; ALP, alkaline phosphatase. aResponse rate according to Paris-I is defined as: ALP ≤3 ×ULN, AST ≤2 ×ULN, and normal bilirubin after 1 year of UDCA therapy.
48
49
3.3.6 Decompensation, HCC, and transplant-free survival
The 10-year incidence rate of hepatic decompensation (ascites, variceal bleeding, or hepatic
encephalopathy, whichever came first) decreased over time: 18.5% in the 1970s, 13.7% in the
1980s, 8.5% in the 1990s, and 5.8% in the 2000s (Figure 3-4Ai). All pairwise comparisons were
significantly different, except the difference between the 1970s and 1980s cohorts (P = 0.45). In
a multivariable Cox regression, a temporal trend of lower decompensation risk was observed
after adjusting for sex and age at diagnosis (Figure 3-4Bi, Table S3-6, P = 0.07). Calendar time
as a continuous variable was a significant predictor for hepatic decompensation (HR per 10-year
increase: 0.57, 95% CI 0.44-0.75, P < 0.001).
The 10-year HCC incidence rates across the investigated decades were: 10.3%, 4.0%, 2.1%, and
2.3%, respectively (Figure 3-4Aii). The Kaplan-Meier estimate of cumulative HCC incidence
was significantly higher in the 1970s compared to the 1980s (P = 0.01), 1990s (P < 0.001), and
2000s (P < 0.001). In a multivariable Cox regression, calendar time was not a significant
predictor for HCC risk (P = 0.68) after adjusting for sex, age at diagnosis, and UDCA treatment
(Figure 3-4Bii, Table S3-7).
The 10-year liver-related death rate decreased from 1970-2009: 34.6%, 13.2%, 5.6%, and 6.4%
(P < 0.001). Furthermore, the 10-year transplant-free survival rate improved over the four
respective investigated decades: 48.4%, 68.7%, 79.7%, and 80.1% (Figure 3-4Aiii). There was a
significant difference in transplant-free survival between the 1970s and 1980s (P < 0.001), and
between the 1980s and 1990s (P < 0.001). However, the transplant-free survival rates between
the 1990s and 2000s were equivalent (P = 0.80). In a multivariable Cox regression, calendar time
remained an independent predictor of transplant-free survival, and earlier decades were
associated with an increased risk for liver transplantation and all-cause mortality (Figure 3-4Biii,
Table S3-8). Furthermore, the 10-year transplant-free survival of PBC patients has improved
even when compared to an age- and gender-matched general population (1970s: HR 4.38, 95%
CI 3.54-5.43, P < 0.001; 1980s: HR 2.90, 95% CI 2.60-3.24, P < 0.001; 1990s: HR 2.14, 95% CI
1.94-2.36, P < 0.001; 2000s: HR 1.93, 95% CI 1.69-2.21, P < 0.001).
50
Figure 3-4. Time-to-event analyses of decompensation, hepatocellular carcinoma (HCC), and
liver transplantation or death over calendar time. A) Kaplan-Meier (crude) and B) Multivariable
Cox regression (adjusted) estimates of i) cumulative incidence of decompensation, ii) cumulative
incidence of hepatocellular carcinoma (HCC), and iii) transplant-free survival.
Follow-up (years)
1086420
Cum
ulat
ive
inci
denc
e of
deco
mpe
nsat
ion
(%)
20
15
10
5
0
1970-1979-censored
2000-20091990-20001980-19891970-1979
Follow-up (years)1086420
Cum
ulat
ive
HC
C
inci
denc
e (%
)
15
10
5
0
1970-1979-censored
2000-20091990-20001980-19891970-1979
Follow-up (years)1086420
Tran
spla
nt-fr
ee
surv
ival
(%)
100
80
60
40
20
0
1970-1979-censored
2000-20091990-20001980-19891970-1979
Follow-up (years)1086420
Cum
ulat
ive
inci
denc
e of
de
com
pens
atio
n (%
)
20
15
10
5
0
Crude AdjustedA) B)i)
ii)
iii)
i)
No. at risk1970-19791980-19891990-19992000-2009
No. at risk1970-19791980-19891990-19992000-2009
No. at risk1970-19791980-19891990-19992000-2009
50492
1039810
46434936559
40367784348
34298637163
57574
11401046
59622
12021127
139790
15201666
112697
14101471
93574
12511121
74496
1110742
61420954451
49336788194
143858
17541815
117763
16411611
99641
14661242
78557
1315829
63472
1101502
51379876224
P < 0.001
P < 0.001
P < 0.001
P = 0.07
Follow-up (years)1086420
Tran
spla
nt-fr
ee
su
rviv
al (%
)
100
80
60
40
20
0
P < 0.001
iii)
Follow-up (years)1086420
Cum
ulat
ive
HC
C
inci
denc
e (%
)
5
4
3
2
1
0
ii)P = 0.68
51
3.4 Discussion In this study of a large, internationally representative cohort of PBC patients, we demonstrate
that patients diagnosed in recent decades are older and have a milder disease stage compared to
patients diagnosed in earlier decades. In addition, more patients respond favourably to UDCA
therapy and have improved transplant-free survival. To the best of our knowledge no previous
study has reported on these PBC trends. These results provide unique insight into the possible
changing natural history of PBC over the last five decades. It is noteworthy to mention that
similar results have been observed in a study from Sweden that included 246 patients diagnosed
with primary sclerosing cholangitis between 1984 and 2004. Bergquist et al. reported an increase
in age at diagnosis and lower frequency of symptoms in patients diagnosed after 1998
(Bergquist, Said, & Broomé, 2007).
Although some of the observed trends could be potentially attributed to more sensitive AMA
tests that detect the disease at an earlier stage, we speculate that any changes in AMA testing
have not had a major impact in the observed temporal trends. The conventional method of AMA
detection is indirect immunofluorescence, yet there has been an increase in ELISA-based assays
and immunoblotting that have led to greater sensitivity and specificity (Oertelt et al., 2007).
These improvements would translate to an increase in the proportion of AMA-positive patients,
however this has remained unchanged.
We demonstrate a 10-year increase in the mean age at diagnosis from 1970 to 2014. A similar
increase has been reported previously in the Canadian PBC population, in which prevalent cases
in 1996 had a median age of 53, whereas prevalent cases in 2002 had a median age of 57 (Myers
et al., 2009). These numbers coincide with the findings from our study, in which the mean age at
diagnosis in the 1990s and 2000s is 52.8 and 55.0 years, respectively. Furthermore, an increased
proportion of patients diagnosed in recent years are over 50 years of age and account for 71.5%
of patients diagnosed on 2010 and beyond. Comparable results were found within the UK-PBC
cohort, in which 75% of patients prevalent between 2008 and 2010 were over 50 years of age
(Carbone et al., 2013).
The increase in age may be attributed to the general aging of the population, as the median age in
Northern America and Europe has reportedly increased from 30 in 1970 to 40 in 2015 (United
52
Nations, Department of Economic and Social Affairs, 2017). This represents a 10-year increase
over a 45-year period, which is similar to the 10-year increase in age at diagnosis we observe
over a 44-year interval. Furthermore, the 34% absolute increase of PBC patients 50 years old and
above from 1970 to 2014 was greater than that of the general population, which was only 11%
(25% in 1970 to 36% in 2015) (United Nations, Department of Economic and Social Affairs,
2017). The increase in age may also be attributed to differences in the trigger for a PBC
diagnosis over the years. Although we are not able to assess the symptoms in our cohort, we
speculate that patients in recent decades are predominantly asymptomatic and are therefore
diagnosed when they see their physician to undergo routine testing of liver function, which
occurs more frequently in older individuals. Conversely, younger patients in earlier decades were
more likely to develop symptoms, which led to their diagnoses (Mahl et al., 1994; M. Prince et
al., 2002). Lastly, the increase in age may be disease-specific and represent a shift in the natural
history of PBC towards a new older at-risk population, considering the increase in age was
observed irrespective of biochemical disease stage. It can also be speculated that the later onset is
a result of a prolonged subclinical disease period and potentially a delayed exposure to an
unknown environmental trigger due to temporal changes in lifestyle.
An older age at diagnosis is clinically important because it has been associated with an increased
likelihood of meeting Paris-I criteria for response to UDCA (Carbone et al., 2013). Similarly, we
found an older age at diagnosis to be an independent predictor of Paris-I response, yet calendar
time was not a significant predictor. These results indicate the increase in age at diagnosis may
be an important factor contributing to the increase in UDCA-response rather than calendar time
itself. Furthermore, the low response rates observed in earlier decades can be a result of
inadequate UDCA dosages and the delay in treatment. The importance of an adequate UDCA
dosage of 13-15mg/kg per day has been emphasized in a study that found 40% of UDCA-non-
responders in whom the dosage was increased became responders (Angulo, Dickson, et al., 1999;
Lammers et al., 2016).
In recent decades, patients present at an older age, yet they have milder biochemical and
histological disease stage. Improved disease severity might be explained by an earlier detection
of PBC due to improved disease awareness leading to liver function tests and AMA assays
(European Association for the Study of the Liver, 2009; M. I. Prince et al., 2004). The
53
histological disease stage at diagnosis has important prognostic implications for UDCA-response
and survival. Advanced histological stages are associated with an increased risk of treatment
failure (Corpechot et al., 2008). In addition, the survival of UDCA-treated patients in stage I/II is
similar to that of an age- and sex-matched control population, while the probability of liver
transplantation or death is significantly increased in patients with advanced histological stages
(Corpechot et al., 2005).
Although a decrease in the number of liver transplantations for PBC has been reported over the
years (Lee et al., 2007), an improvement in transplant-free survival has not been previously
documented. In a Canadian population-based study of patients diagnosed between 1996 and
2002, Myers et al. did not observe a significant difference in survival according to year of
diagnosis (Myers et al., 2009). The lack of difference in survival may be attributed to the small
interval of study, which only spanned six years. The reported increase in median age of the
general population well reflects an increase in life expectancy over time (United Nations,
Department of Economic and Social Affairs, 2017); therefore transplant-free survival was
compared to that of the general population. Our study showed that transplant-free survival
improved over a 44-year period, even when compared to the general population, and supports its
potential role in the increased prevalence of PBC.
The inclusion of a large population of PBC patients from different geographical regions, long-
term follow-up, and broad study period are some of the strengths of our study. However, some
limitations need to be considered. First, the 1970s and 1980s cohorts were susceptible to a delay
in documentation since study entry can be many years after the date of diagnosis in these
cohorts. As such, the difference in years between these two dates was included in all
multivariable analyses and we assessed a sub-group of patients with a maximum two-year
difference. The same trends emerged in the sub-group analyses, thus excluding the possibility
that the delay in documentation is the reason for an advanced disease in the early cohorts.
Second, due to the retrospective nature of the study, biochemical data was not available for all
patients and thus response to UDCA could not be determined for all patients. To account for
missing laboratory values, all analyses were repeated in an imputed dataset and revealed similar
results. Lastly, the trends observed in our study cohort could not be assessed for correlations with
symptom profiles or various environmental factors previously associated with PBC, such as
54
smoking, age at first pregnancy, or the use of hormonal replacement therapy (Gershwin et al.,
2005). Even though the trends observed may be due to a selection of patients whose diagnosis is
triggered by symptoms or complications in earlier decades rather than routine liver function tests
as in recent decades, we describe the presenting characteristics of a typical PBC patient seen by
physicians and how they have changed over time. The observed temporal trends warrant further
investigation in other PBC populations to determine whether they are universally applicable and
to explore the potential influence of a changing environmental trigger.
In conclusion, we demonstrate a 10-year increase in age at diagnosis accompanied by milder
disease severity at presentation of PBC patients. These findings provide the most comprehensive
evidence of a changing natural history of PBC to date.
3.5 Supplementary Tables and Figures Table S3-1. Distribution of PBC patients across calendar time and center
Data represented as n (% within corresponding decade). ᵃComprised of centers across the Netherlands (mainly secondary centers). bTertiary center. cSecondary center. dComprised of two centers.
57
Table S3-2. Calendar time trends in patients with a maximum lag of 2 years between diagnosis and study entry
Characteristics 1970-1979 (n=20)
1980-1989 (n=245)
1990-1999 (n=1331)
2000-2009 (n=1687)
≥2010 (n=235)
P-value
Age at diagnosis, ya 49.3 (12.9) 52.3 (11.7) 52.9 (11.6) 55.0 (12.6) 57.0 (12.1) ˂0.001
10-year liver-related death 53.2 14.0 4.9 6.5 - <0.001
Data represented as mean (standard deviation), n (%), or median (interquartile range). AMA, antimitochondrial antibody; ALP, alkaline phosphatase; ULN, upper limit of normal; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LLN, lower limit of normal; APRI, AST to platelet ratio index; UDCA, ursodeoxycholic acid; HCC, hepatocellular carcinoma. aAge at diagnosis not available for one patient in 2000-2009 cohort. bAMA status was available for 3430 (97.5%) patients. cALP, bilirubin, AST, and ALT were log transformed prior to analyses and availability for laboratory values is as follows: ALP: 2662 (75.7%); Bilirubin: 2586 (73.5%); AST: 2593 (73.7%); ALT: 2271 (64.6%); Albumin: 2123 (60.3%); Platelet count: 1998 (56.8%) dThe cut-point APRI >0.54 at baseline is predictive of liver transplantation or death (Trivedi et al., 2014). eBiochemical disease stage classification according to Rotterdam criteria (E. M. Kuiper et al., 2009) was available in 2057 (58.5%) patients.
59 fUDCA therapy status was available for 3452 patients (98.1%). gUDCA dosage was available for 1319 (43.6%) of UDCA-treated patients. hResponse was determined based on the availability of laboratory values at 1 year of UDCA therapy. Response according to Toronto criteria was calculated after 2 years of UDCA therapy.
60
Figure S3-1. Mean age at diagnosis over calendar time stratified by A) Center (each line corresponds to an individual center); B) Sex; and
Data represented as n (%). UDCA, ursodeoxycholic acid. ᵃResponse was determined based on the availability of laboratory values at 1 year of UDCA therapy. Response according to Toronto criteria was calculated after 2 years of UDCA therapy. bResponse according to the GLOBE score was established when the calculated value did not surpass the age-specific threshold (Lammers et al., 2015).
64
Table S3-5. Response rate over calendar time in UDCA-treated patients who did not meet criteria at baseline
Data represented as n (%). UDCA, ursodeoxycholic acid. ᵃResponse was determined based on the availability of laboratory values at 1 year of UDCA therapy. Response according to Toronto criteria was calculated after 2 years of UDCA therapy.
65
Table S3-6. Multivariable Cox regression of 10-year hepatic decompensation (n=2962)
Variable HR 95% CI P value
Year of diagnosis 0.07
1970-1979 1.00
1980-1989 1.32 0.61-2.87 0.48
1990-1999 1.11 0.46-2.70 0.81
2000-2009 0.73 0.28-1.87 0.51
Male sex 1.25 0.80-1.98 0.33
Age at diagnosis (years) 0.21
<30 1.00
30-39 1.35 0.40-4.50 0.63
40-49 1.84 0.58-5.87 0.30
50-59 1.82 0.57-5.82 0.31
60-69 2.00 0.61-6.61 0.25
>70 3.09 0.88-10.81 0.08
Difference between diagnosis and study entry (years) 1.08 1.04-1.13 <0.001
HR, Hazard ratio; CI, confidence interval.
66
Table S3-7. Multivariable Cox regression for 10-year HCC incidence (n=3963)
Variable HR 95% CI P value
Male sex 3.48 2.05-5.89 <0.001
UDCA 0.38 0.21-0.67 0.001
Year of diagnosis 0.68
1970-1979 1.00
1980-1989 1.19 0.49-2.89 0.70
1990-1999 1.51 0.51-4.48 0.46
2000-2009 1.88 0.58-6.06 0.29
Age at diagnosis (years) 0.11
30-39 1.00
40-49 0.27 0.10-0.75 0.01
50-59 0.53 0.28-1.02 0.06
60-69 1.63 0.34-1.15 0.13
≥70 0.62 0.23-1.68 0.35
Difference between diagnosis and study entry (years) 1.19 1.13-1.26 <0.001
Total bilirubin, ×ULN 0.53 (0.40-0.71) 0.50 (0.38-0.68)
ALP, ×ULN 1.84 (1.20-3.03) 1.25 (0.88-1.96)
Albumin, ×LLN 1.17 (1.09-1.26) 1.17 (1.09-1.26)
AST, ×ULN 1.20 (0.83-1.80) 0.84 (0.64-1.18)
ALT, ×ULN 1.45 (0.93-2.30) 0.83 (0.57-1.31)
Platelet count, 109/L 252 (202-304) 247 (197-300)
Bilirubin ULN (mg/dL), median (IQR)d 1.0 (1.0-1.2) 1.0 (1.0-1.2)
PBC, primary biliary cholangitis; IQR, interquartile range; SD, standard deviation; AMA, antimitochondrial antibody; UDCA, ursodeoxycholic acid; ULN, upper limit of normal; ALP, alkaline phosphatase; AST, aspartate aminotransferase; ALT, alanine aminotransferase. aHistological disease stage at study entry available for 1485 patients (53.1%)- stage I/II: 1111 patients (74.8%), stage III/IV: 374 patients (25.2%). bBiochemical disease stage at study entry available for 2259 patients (80.8%) – mild: 2094 (74.9%), moderate: 165 (5.9%), advanced: 0 patients. cLaboratory parameters other than bilirubin were not available for all patients:
Time zero cohort: ALP (n=2627), albumin (n=2259), AST (n=2531), ALT (n=2134), platelet count (n=2039). 1-year cohort: ALP (n=2824), albumin (n=2043), AST (n=2542), ALT (n=2309), plateletcount (n=1370).
dThe upper limit of normal for bilirubin was variable per center.
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4.3.2 Normal bilirubin quartiles are associated with liver transplant-free survival
The quartiles in each individual cohort were formulated according to the following bilirubin
levels (median [IQR], ×ULN): 0.53 (0.40-0.71) and 0.50 (0.38-0.68), respectively. In Kaplan-
Meier analysis of patients that had normal bilirubin at time zero, the cumulative 10-year liver
transplant-free survival rate decreased with higher bilirubin quartiles and was 92.4%, 89.0%,
87.9%, 77.5% from quartiles 1-4 (Q1-Q4), respectively (Figure 4-1). In pairwise comparisons,
Q4 was significantly different from Q1-Q3 (all P < 0.0001). Similar results were obtained in the
Kaplan-Meier analysis of the 1-year cohort, in which the 10-year liver transplant-free survival
rates with increasing bilirubin quartiles were 91.3%, 91.4%, 86.2%, and 76.7%. Q3 and Q4 were
significantly different from one another and from the remaining quartiles (all P < 0.05). In
multivariable Cox regression analyses, normal bilirubin quartiles were a significant predictor for
transplant-free survival. In the time zero cohort, the risk for liver transplantation or death
increased with higher bilirubin quartiles: Q1 (reference), Q2 (HR 1.24, 95% CI 0.85-1.81, P =
0.26), Q3 (HR 1.28, 95% CI 0.90-1.83, P = 0.18), Q4 (HR 2.16, 95% CI 1.53-3.04, P < 0.0001).
A similar trend was observed in the 1-year cohort: Q1 (reference), Q2 (HR 0.98, 95% CI 0.68-
1.41, P = 0.91), Q3 (HR 1.43, 95% CI 1.01-2.01, P = 0.04), Q4 (HR 2.27, 95% CI 1.65-3.12, P <
0.0001).
Figure 4-1. Transplant-free survival of the normal bilirubin quartiles in patients with normal
HR, hazard ratio; CI, confidence interval; ULN, upper limit of normal; UDCA, ursodeoxycholic acid; ALP, alkaline phosphatase. aHistological stage determined at study entry.
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Figure S4-2. The association between bilirubin levels (mg/dL) and risk for liver transplantation
or death. Hazard ratios and 95% CI were estimated by a restricted cubic spline function in A) the
time zero cohort and B) the 1-year cohort. The bilirubin reference in each cohort is 0.65 mg/dL
and the test for curvature is significant in both cohorts (P < 0.0001 and P = 0.04).
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Figure S4-3. The association between bilirubin levels (×ULN) and risk for liver transplantation
(LT) or death. Hazard ratios and 95% CI were estimated by a restricted cubic spline function at
2-4 years. The bilirubin reference in each cohort is 0.6×ULN.
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Figure S4-4. The distribution of patients with bilirubin below and above 0.6×ULN in those with
normal bilirubin at baseline (n=2791).
Year of diagnosis
Perc
enta
ge o
f pat
ient
s
1970-19791980-19891990-19992000-2009 ≥20100
20
40
60
80
100
≤0.6×ULN
>0.6×ULN
Baseline bilirubin
P < 0.001
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Chapter 5
General Discussion 5
5.1 Calendar Time Trends
5.1.1 Discussion
Insight into the temporal changes of clinical features at presentation of PBC patients has been
limited. Studies suggest that patients diagnosed in recent decades are predominantly
asymptomatic (T. R. Baldursdottir et al., 2012; Floreani et al., 2011). There are also reports of an
increase in age at diagnosis, and no difference in the female: male ratios (Floreani et al., 2011;
Rautiainen et al., 2007; Sakauchi et al., 2007). Whether there is a difference in the survival rates
of patients according to year of diagnosis is inconclusive. One study from Finland reported
improved survival and a Canadian study did not find year of diagnosis to be a significant
predictor for survival (Myers et al., 2009; Rautiainen et al., 2007).
We sought to describe patient and disease characteristics over a 44-year period in a globally
representative population. This study contains the most comprehensive evidence to date due to
its size, long-term follow-up, and the inclusion of multiple centers worldwide. The main findings
from this retrospective study indicated that the mean age at diagnosis increased by 2-3 years per
decade from 47 years to 57 years, the female to male ratio and AMA-positivity were unchanged,
the proportion of patients presenting with mild biochemical and histological disease stage
increased, more patients responded to UDCA, and there were lower decompensation rates and
higher transplant-free survival rates in more recent decades. These results support the hypothesis
that the natural of history of PBC has changed over time. The most plausible contributors to the
observed changes in the natural history of PBC are improved awareness, early diagnosis, and
availability of treatment. However, the possibility that these changes may also be due to
environmental factors cannot be excluded since multiple studies have indicated that the incidence
of PBC is increasing in various geographical regions (Al-Harthy & Kumagi, 2012; Floreani et
al., 2011).
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5.1.1.1 Symptoms
A key difference in patients diagnosed in recent times compared to those diagnosed in earlier
times is symptomology, as previous studies have consistently reported a decrease in the
proportion of symptomatic patients (T. R. Baldursdottir et al., 2012; Floreani et al., 2011).
Symptoms are an important aspect to consider when assessing these temporal trends, as studies
show that asymptomatic PBC patients present with an earlier histological stage and improved
biochemical measures (Mitchison et al., 1990). Furthermore, symptomatic presentation may
predict an inadequate response to UDCA and poor prognosis (Jones, Al-Rifai, Frith, Patanwala,
& Newton, 2010; Quarneti et al., 2015). Although asymptomatic PBC is generally less severe at
diagnosis than symptomatic, being asymptomatic is not always synonymous with having an early
disease (Kumagi & Heathcote, 2008). Unfortunately, an evaluation of the changes in
symptomology was not possible because this information was not available in the GLOBAL
PBC database. Since most patients nowadays present without symptoms, we speculate that
patients diagnosed in recent decades from our cohort were predominantly asymptomatic.
5.1.1.2 AMA testing and diagnosis
One of the current diagnostic criteria for PBC is seropositivity for AMA. The conventional
method for AMA detection is IIF, yet there has been an increase in ELISA-based assays and
immunoblotting due to the identification of specific antigen reactivity (Oertelt et al., 2007).
These improvements have led to greater sensitivity and specificity when detecting AMA.
However, there is still heterogeneity in the results and antigenic epitopes tested across
laboratories. Furthermore, patients are generally screened for AMA after they demonstrate
cholestatic laboratory results, such as elevated ALP or GGT. Improvements in the sensitivity of
AMA tests would hypothetically translate to an increase in the proportion of AMA-positive
patients, however this has remained unchanged in our cohort. Therefore, we speculate that any
changes in the techniques used for AMA testing have not played a major role in the distinct
presentation of patients over calendar time. A significant role on the observed changes is
expected to result from increased physician awareness and routine testing of liver function in
recent years, which suggests that earlier cohorts were largely composed of patients that were
diagnosed due to symptoms/complications and only a minority of patients’ diagnosis were
prompted by routine testing.
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5.1.1.3 Age relative to the general population
In line with our findings, there have been reports that the age at diagnosis has increased over
time in Italian and Japanese populations (Floreani et al., 2011; Sakauchi et al., 2007). Increased
routine testing in recent years may account for the increase in older patients being diagnosed
with PBC, as screening occurs more frequently in the elderly (Spalding & Sebesta, 2008). The
increase in age at diagnosis may potentially be associated with the increase in age seen in the
general population, therefore it is critical that the age data be compared to that of the general
population. In the PBC population, there was a gradual increase in the proportion of patients 50
years old and above at the time of diagnosis from 38% in the 1970s to 72% after 2010, an overall
increase of 34% in the investigated time frame. In the general population there has been an
overall increase of 11% in the proportion of individuals ≥50 years old, which increased from
25% in 1970 to 36% in 2015 (United Nations, Department of Economic and Social Affairs,
2017). When comparing age trends in PBC patients to that of the general population, the
percentage of patients ≥50 years old are overall higher in the PBC population, which may reflect
the restricted age spectrum observed in PBC since patients under 18 years old were excluded and
PBC typically affects middle-aged individuals. It is logical that there are higher proportions of
this age group in the PBC cohort compared to that of the general population. Yet, the increase of
this age group in the PBC population was greater compared to that of the general population
within the specified time frame. This suggests that there may be additional factors contributing to
the increase in age observed in PBC other than an aging population. Additional potential
contributors to the increase in age include a delayed trigger for PBC as a result of changes in
environmental factors or a longer incubation period from when there is AMA-positivity to the
development of abnormal liver biochemistry.
5.1.1.4 Biochemical and histological disease stage
Liver biochemistry and histological findings in the GLOBAL PBC cohort indicated that patients
presented at an earlier disease stage, which can also be attributed to improved patient care and an
earlier diagnosis of PBC. A study from Iceland compared the proportion of patients who
presented with an advanced histological stage (III-IV) in 1991-2000 to those from 2001-2010
and reported no difference over time, accounting for 28% of patients (T. R. Baldursdottir et al.,
2012). Interestingly, they did note a decrease in the proportion of asymptomatic patients, which
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further supports the notion that symptoms may not directly correlate with histology. In contrast,
we noted patients with an advanced histological stage (III-IV) accounted for 33% in 1990-1999
and that there was a decrease to 23.5% in 2000-2014.
5.1.1.5 Treatment regimen with UDCA
The availability and timing of treatment for PBC can have a major impact on response to
treatment and the subsequent development of clinical outcomes. Although UDCA was used as
off-label therapy starting in the late 1980s, it did not gain FDA approval until 1997. UDCA is
the standard treatment for PBC because it has been shown to delay histological progression and
improve survival (Parés et al., 2000; Shi et al., 2006). The introduction of UDCA was likely a
major contributor for the decrease in the number of liver transplantations for PBC, compared to
the unchanged number of liver transplantations for PSC, a similar chronic disease to PBC for
which there is no available treatment (Lee et al., 2007). Even when UDCA was available for the
treatment of PBC, it seems that the treatment regimen was not optimal, as inappropriate dosages
were administered in earlier decades that deviated from the currently recommended dosage of
13-15mg/kg/day.
Another aspect in the treatment regimen of PBC that has greatly changed over time is timing to
treatment. Although there was a substantial portion (88%) of patients who were diagnosed in the
1980s that received UDCA, they still experienced worse response rates and transplant-free
survival rates compared to patients diagnosed in more recent decades. Time to treatment was a
critical factor that influenced their outcomes since they received UDCA many years after their
diagnosis. In our analysis, the time from diagnosis to study entry was a significant predictor for
hepatic decompensation, HCC development, and transplant-free survival. In the time between
their diagnosis and the start of treatment, the disease had an opportunity to progress and therefore
the introduction of UDCA at this more advanced disease stage would not be as beneficial had it
been administered at an earlier disease stage (Parés et al., 2000; Shi et al., 2006). The time lag to
treatment administration is the greatest in the earlier cohorts and is likely attributable to the lack
of availability of UDCA. Overall, this suggests that the treatment regimen of PBC with UDCA
has improved considerably by the usage of more appropriate dosages and a prompt initiation
after diagnosis.
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5.1.1.6 Clinical outcomes
As mentioned, there has been an increase in median age and the proportion of individuals ≥50
years old in the general population, which reflects an increase in life expectancy (median life)
over time. We showed that transplant-free survival increased over calendar time in PBC patients,
as is also expected in the general population. Therefore, we compared transplant-free survival
over time with respect to an age-, gender-, and birth year-matched general population. These
results indicated that the survival of PBC patients has improved over time even when compared
to the general population. An increase in survival over calendar time, especially when compared
to the general population, has not been previously shown in a globally representative population.
A Canadian study failed to show an improvement in survival from 1996 to 2002 most likely due
to the short period analyzed (Myers et al., 2009). A study from Finland that evaluated survival
from 1988 to 1999 reported improved survival, in which the age-, gender- and study area-
adjusted HR for all-cause mortality was 0.6 per a 10-year increase in year (Rautiainen et al.,
2007). An improved transplant-free survival may be potentially associated with the strongly
documented increase in the prevalence of in various geographical regions.
There was no difference in HCC incidence after the 1980s, however there was a decrease in
decompensation over time. This can be attributed the overall low incidence of HCC when
compared to decompensation. Therefore, one is unable to capture any differences due to the
small number of events.
5.1.1.7 Trends in primary sclerosing cholangitis
It is relevant to assess how the natural history of another cholestatic liver disease may have
evolved over time as compared to PBC. A Swedish study compared the clinical presentation of
patients with PSC between 1984 and 2004 with an emphasis on patients diagnosed before 1998
and after 1998. In the later cohort of patients diagnosed after 1998, they found an older age at
diagnosis (41 years old vs 37 years old), a lower frequency of symptoms (47% vs 63%), and a
lower rate of inflammatory bowel disease (IBD) (Bergquist et al., 2007). The lower age in the
earlier cohort was partially attributed to the higher proportion of patients with IBD, since these
patients undergo regular clinical check-ups. These results suggest that an increase in age may not
be specific to PBC and that it may also be applicable to other liver diseases. Furthermore, it
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indicates that overall health care has improved, and patients with various liver diseases are being
diagnosed at an earlier disease stage without symptoms. A key difference between these two
diseases is that PBC exhibits improved outcomes relative to PSC, as suggested by the stable
number in liver transplantations for PSC, compared to a decrease in liver transplantations for
PBC.
5.1.2 Strengths and limitations
5.1.2.1 Strengths
There are strengths associated with this study that entail the population in which it was
performed, and the methodology used. The first strength of the GLOBAL PBC cohort is that it is
a multicenter cohort that includes centers through Europe and North America. This makes the
study unique from other studies that have also evaluated changes in patient characteristic because
the changes can be extrapolated to various geographical regions, rather than be limited to a
certain geographical region. Furthermore, due to the inclusion of patients dating back to the
1960s and their long-term follow-up, an analysis of a broad time period was possible, as well as
their respective rates of complications and outcomes. The 44-year period that was assessed is not
only the longest time period assessed for such a descriptive study, but it also spans the time prior
to UDCA and after UDCA, which allowed us to gain insight into how the introduction of this
treatment affected its natural history.
The survival of the general population has undoubtedly increased over time. Therefore, a simple
description of changes over time would not have been sufficient because it is expected that
survival has improved over time. Therefore, it was critical for the survival to be compared to that
of the general population to adequately assess whether survival indeed changed over time.
5.1.2.2 Limitations
Most epidemiological studies of PBC have been conducted in patients from Western countries
and less so in other populations, such as Asian or Hispanic populations. One of the limitations of
this study is also the limited ethnicity of the population, as the only centers included were from
Europe and North America. Therefore, it is still uncertain whether these changes are applicable
to other ethnic populations or from other geographical locations, especially in populations that
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have been reported to have a more severe disease stage, such as African American and Hispanic
populations (Peters et al., 2007). Similar results are to be expected, however, as a study on the
temporal trends of a symptomatic Japanese population also reported an increase in age and
improved liver biochemistry at study entry (Sakauchi et al., 2007).
Another limitation of this study is that the association of the observed changes could not be
correlated to symptoms or other environmental factors that have been previously reported to be
associated with PBC, such as smoking, UTIs, and hormonal replacement therapy because these
variables were absent from the GLOBAL PBC database. It would be of relevance to assess their
potential association because it is possible that evolving habits in terms of these environmental
factors may have contributed to a delay in PBC presentation and its associated milder disease
severity.
5.1.3 Implications
We have provided a description of the presenting disease characteristics of a typical PBC patient
seen by physicians during a specified time frame. This in itself is useful because it proves that
the disease is not static and has evolved over time. It also documents the influence that UDCA
has had on the natural history of PBC, as well as the improved health care over time. This is
reassurance that the improvement of care for patients with PBC through an early diagnosis and
timely access to treatment can translate into improved outcomes for patients. Furthermore, it
emphasizes that there are various factors affecting the outcome in patients, including diagnosis
year, availability to treatment, time to treatment, dosage of UDCA, as well as baseline disease
characteristics.
As the disease has proven to not be static and evolving, the prognostic parameters used in the
clinic should be in line with the patients of today. For example, the Mayo model was developed
in a time when patients were at a symptomatic and advanced stage without available treatment.
This model is not applicable in the PBC population of today and has been found to underestimate
the survival of patients.
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Bilirubin is one of the major predictors for prognosis, it is included in various prognostic models
and response criteria, and the number of patients presenting with normal bilirubin increased over
time, thus this biochemical parameter was further evaluated in the second study.
5.2 Bilirubin Within the Normal Range
5.2.1 Discussion
Up to date, the majority of studies have made an emphasis on normal bilirubin, as indicated by
the inclusion of this criterion for various response criteria, such as Paris-I, Paris-II, and
Rotterdam. Indeed, a study that evaluated various thresholds of bilirubin reported that the best
threshold for predicting liver transplantation or death was at the ULN but did not assess
thresholds below the ULN (Lammers et al., 2014). Bilirubin is not abnormal until later stages of
disease and thus, the predictive value of bilirubin is thought to be limited during early stages of
disease to a small number of patients. In our first study, we showed that there has been an
increasing proportion of patients that have normal bilirubin at study entry, and presumably an
even higher number after 1 year of treatment because of the effect of UDCA on bilirubin. A
recent clinical trial on OCA showed that bilirubin decreased even within the normal range and it
is unknown whether this translates to an improved outcome. We sought to determine whether
bilirubin levels below the ULN could be predictive of transplant-free survival.
When we evaluated the predictive value of bilirubin below the ULN, a bilirubin threshold of
0.6×ULN was established as the optimal threshold. Patients with bilirubin levels below this
threshold possess the lowest risk for liver transplantation or death, whereas the risk increases
linearly above this threshold. Furthermore, we show that in patients who are above the threshold
and subsequently experience a decrease in bilirubin to below the threshold as a result of
treatment with UDCA, there is an improvement in transplant-free survival. It was also suggested
that increasing bilirubin levels over time above this threshold may indicate the future
development of a poor clinical outcome.
5.2.1.1 Sensitivity of ULN of bilirubin for predicting outcome
Our results indicate that the current ULN for bilirubin may not be a sensitive indicator of risk in
PBC. One potential reason for this finding is that PBC is a female predominant disease, yet the
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ULN is determined in the general population, whereby male bilirubin values may skew the ULN
as they tend to have higher bilirubin levels than females. Furthermore, we speculate that it may
have to do with the relative increase in bilirubin required to reach the ULN, given the modal
bilirubin level in a healthy population was 0.4mg/dL (Zucker et al., 2004). The ULN is usually
between 1-1.2mg/dL, which suggests an increase of 0.6-0.8mgdL would be required.
5.2.1.2 Bilirubin in the general population
There is a positive relationship with bilirubin and the risk for liver transplantation and death
beyond the 0.6× ULN threshold in PBC. Interestingly, there appears to be a different relationship
with bilirubin in individuals without liver disease, in whom increased bilirubin levels have been
associated with a decreased incidence of cardiovascular, rectal cancer, and diabetes (Levitt &
Levitt, 2014; Zucker et al., 2004). In an NHANES study of 4303 individuals 60 years old and
above from 1999 to 2004, individuals with bilirubin between 0.1-0.4mg/dL had the highest
mortality rates. In comparison to those with bilirubin of 0.5-0.7mg/dL, they had a 1.36-fold
increase in risk. Although individuals with bilirubin ≥0.8mg/dL also had higher mortality rates, it
was not statistically significant (Ong et al., 2014). The anti-inflammatory and antioxidant
properties of bilirubin may account for these differences. Furthermore, it is important to keep in
mind that total bilirubin is primarily composed of unconjugated bilirubin in these individuals as
compared to PBC patients, in which elevated bilirubin would be primarily composed of
conjugated bilirubin.
5.2.1.3 Risk stratification in PBC
There are various liver biochemistry parameters that may be utilized in PBC for risk stratification
of which ALP and bilirubin are the primary parameters (Lammers et al., 2014). These are readily
available as they are usually routinely checked in patients, and thus represent a primary means
for risk stratification. However, established response criteria rely on these biochemical
parameters in a dichotomized form, which may lead to a loss in predictive ability. Therefore, risk
stratification in PBC has recently shifted towards prognostic models that utilize various liver
biochemistries to predict transplant-free survival such as the GLOBE score (Lammers et al.,
2015). This score was shown to be superior to other response criteria, potentially because
bilirubin in addition to the other biochemical variables included were input into this model as a
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continuous variable and it includes a variety of prognostic variables for PBC. Response criteria
in which normal bilirubin was a component would lose the increased risk imposed on patients
with normal bilirubin whose levels were above 0.6×ULN.
Biochemical parameters and prognostic models that rely on these biochemical parameters are the
preferred method for risk stratification as compared to liver biopsies. Although histological
staging provides important prognostic information, it is no longer routinely performed due to its
invasive nature. Transient elastography is a promising tool to assess liver fibrosis by measuring
liver stiffness, yet this may not be the best manner to assess response to therapy after a short
period of time, as changes in fibrosis may not be observed until longer follow-up takes place.
Therefore, liver biochemistry parameters represent an important manner of assessing prognosis
as they are easily attainable and can provide long-term prognostic information (Lammers et al.,
2015).
5.2.2 Strengths and limitations
5.2.2.1 Strengths
The strengths of this study include the large number of patients from a globally representative
population and long-term follow-up. In terms of methodology, the predictive ability of bilirubin
was not only assessed at baseline but also up to 4 years of follow-up, which confirms that the
bilirubin threshold can be applied at various independent time points. This is important because
it allows the implementation of the threshold before treatment, and after therapy to assess
whether the patient requires additional therapy. Indeed, we showed that a reduction in bilirubin
by treatment with UDCA can translate into an improved prognosis. The applicability of the
threshold was found in multiple subgroups, stratified according to age, histological stage, ALP,
and UDCA treatment, which emphasizes that the threshold is generalizable. Although the
threshold was not statistically significant in males, we attribute it to a lack of power due to the
small sample size.
Another strength of this study is that the risk imposed by bilirubin levels above 0.6×ULN was
interpreted relative to that associated with bilirubin above the ULN. This allowed us to
demonstrate that there was a positive relationship between bilirubin and the risk for liver
transplantation or death after 0.6×ULN that extended beyond the ULN. We also underline that
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there is not only an increased risk for liver transplantation or death above 0.6×ULN, but that
the events are primarily liver-related in this range. Additionally, we were able to assess trends
in bilirubin over 5 years and its relationship with clinical events due to the imputation of
bilirubin. Imputation is a way to deal with missing data by replacing the missing values with an
estimate that is then analyzed as if they were observed values. It provides an unbiased way to
deal with missing data for analysis.
5.2.2.2 Limitations
The study has some limitations. In PBC, conjugated bilirubin is the form that is mainly elevated
and thus it would be beneficial to measure this type of bilirubin to directly measure the effect of
cholestasis on bilirubin levels (Levitt & Levitt, 2014). However, conjugated bilirubin was not
available and only total bilirubin was available in the GLOBAL PBC database. This is due to
the fact that clinical laboratories can only measure direct and indirect bilirubin through the
diazo reaction, in which direct bilirubin does not only include conjugated bilirubin but also
delta bilirubin. Therefore, in order to measure the conjugated bilirubin fraction separately, one
would need to implement other methods, such as high/performance liquid chromatography or
direct spectrophotometry, which are not ideal for routine use.
Furthermore, bilirubin was assessed relative to the ULN of each center, which was variable
across each center. Patients from centers that have a higher ULN may have accounted for the
increased events observed above 0.6×ULN. However, this limitation was overcome by
excluding those with an ULN above 1.2mg/dL and assessing crude bilirubin levels measured in
mg/dL.
5.2.3 Implications
Since patients with bilirubin between 0.6×ULN and 1×ULN are already at an increased risk for
liver transplantation or death, future intervention studies that assess the benefit of therapeutic
drugs may also include these patients since they may be able to benefit from additional therapies.
One of the clinical trials for OCA specified that patients must either have ALP≥1.67×ULN or
abnormal bilirubin (up to 2×ULN) to be included in the study (Nevens et al., 2016). Whereas, the
BEZURSO trial of bezafibrate adjuvant to UDCA indicated that patients included must be non-
responders to Paris-II criteria which also includes abnormal bilirubin (Corpechot et al., 2017).
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We believe that patients whose bilirubin levels are above the threshold of 0.6×ULN may still
benefit from these therapeutic trials, and if the threshold is implemented, it can lead to an
increase in the number of patients that will become eligible for second-line therapies. This is of
relevance because only a minority of patients that are included in these trials have an abnormal
bilirubin. For example, in the OCA trial, 6% of patients had an abnormal bilirubin in the 5-10mg
group and 10% in 10mg group of OCA (Nevens et al., 2016). Our findings also give insight into
the implications of changes in bilirubin seen in these trials. Depending on the randomization
group, patients included had a mean bilirubin level of 0.69mg/dL (placebo), 0.60mg/dL (OCA
5-10mg), and 0.66mg/dL (OCA 10mg) at baseline. Patients randomized to receive OCA
experienced decreases in bilirubin, whereas the placebo group experienced an increase. Our
results suggest that decreases in bilirubin in the range from 1×ULN to 0.6×ULN are still
beneficial irrespective of whether patients attain the 0.6×ULN threshold. Additionally, in the
BEZURSO trial, mean bilirubin at study entry was 0.8mg/dL after which the median decrease in
patients receiving bezafibrate and UDCA after 2 years was 14% (Corpechot et al., 2017). In
light of these results from clinical trials assessing the benefit of second-line therapies for PBC,
we believe that our study grants insight into how bilirubin changes within the normal range
observed as a result of treatment correlate with transplant-free survival that goes beyond a
normalization of bilirubin being associated with improved prognosis.
Our study also has the implication of early determination of patients that may be at risk for a
future outcome. Currently, patients are not considered at risk if their bilirubin is normal, and thus
may be overlooked until it surpasses the normal range. This emphasizes that patients who are
above the 0.6×ULN threshold but experience gradual increases in bilirubin that remain within the
normal range may be at risk for liver transplantation or death and should be closely monitored.
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Chapter 6
Conclusions 6The natural history of PBC has proven to be evolving over time and patients diagnosed in recent
decades have an increased age at diagnosis, a predominantly mild disease stage at presentation,
and improved clinical outcomes. Furthermore, we established that patients with bilirubin
≤0.6×ULN have the lowest risk for liver transplantation or death, from which point onward there
is a linear increase in risk.
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Chapter 7
Future Directions 7Our study provides the most comprehensive evidence to date for a changing natural history of
PBC. However, there are still some questions that need exploration. One potential research
question that can be addressed is whether the changes observed in this population are also
observed in other populations, such as African Americans and Hispanics, in whom PBC tends to
have a more advanced disease stage. Furthermore, the increase in age of patients is peculiar
given that they present with an earlier disease stage. Therefore, the potential influence of
changing environmental factors on the increase in age can be explored in future studies, such as
smoking.
The natural history of PBC has changed primarily due to the introduction of UDCA as the
majority of changes were observed thereafter. The introduction of a new treatment for PBC may
drive further changes. Therefore, it would be interesting to determine how the natural history of
PBC may change in the future with the introduction of OCA or other therapeutic agents and
whether it may impose greater improvements on transplant-free survival.
Bilirubin was found to be an independent predictor of transplant-free survival even within the
normal range. The fact that the normalization of bilirubin is not associated with an absence of
risk raises the question of whether the same may be true for other biochemical parameters such
as ALP. A future study may assess the predictive value of ALP below 1.67×ULN in UDCA-
treated patients, as current studies use this threshold for inclusion in clinical trials and as part of
the primary endpoint. An elevation of ALP is necessary for a diagnosis of PBC, therefore its
predictive ability below 1.67×ULN need be assessed starting after 1 year of treatment, at which
point decreases in ALP are observed.
It may be that bilirubin imposes different risks in the general population compared to PBC
patients. In the general population, it seems that low conjugated bilirubin levels are associated
with an increased risk for mortality. Therefore, it would be of interest to compare mortality of
patients with PBC to that of the general population while adjusting for bilirubin and stratifying
conjugated and unconjugated bilirubin to gain further insight into the role of bilirubin in PBC.
105
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Copyright Acknowledgements
JOHN WILEY AND SONS LICENSE TERMS AND CONDITIONS
Jul 03, 2018
This Agreement between Ms. Carla Fiorella Murillo Perez ("You") and John Wiley and Sons ("John Wiley and Sons") consists of your license details and the terms and conditions provided by John Wiley and Sons and Copyright Clearance Center.
License Number 4353830876851
License date May 21, 2018
Licensed Content Publisher
John Wiley and Sons
Licensed Content Publication
Hepatology
Licensed Content Title Milder disease stage in patients with primary biliary cholangitis over a 44‐year period: A changing natural history
Licensed Content Author Carla F. Murillo Perez, Jorn C. Goet, Willem J. Lammers, et al
Licensed Content Date Apr 6, 2018
Licensed Content Volume 67
Licensed Content Issue 5
Licensed Content Pages 11
Type of use Dissertation/Thesis
Requestor type Author of this Wiley article
Format Print and electronic
Portion Full article
Will you be translating? No
Title of your thesis / dissertation
The changing natural history of primary biliary cholangitis and its influence on risk stratification
Expected completion date Sep 2018
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110
Publisher Tax ID EU826007151
125
Appendix
Figure. Flow chart depicting patient exclusion criteria for the GLOBAL PBC cohort.
GLOBAL PBCcohortN=6677
AIH or otherconcomintant liver
diseasen=193
Uncertain diagnosisn=44
No laboratory valuesn=3
Unknown follow-upor short follow-up
n=1068Short treatment
with UDCAn=268
Total patientsn=5101
126
Contributions Carla Fiorella Murillo Perez had access to the data and performed the majority of the statistical
analysis. Harry Janssen, Jordan Feld, and Bettina Hansen provided guidance throughout the
course of my research, from study design to the interpretation of results. Bettina Hansen also
provided assistance with statistical analyses. Jorn Goet, Willem Lammers, Henk van Buuren,
Maren Harms, and Adrian van der Meer provided feedback and aided with critical revision of the
study for important intellectual content.
Acquisition of data was a collective effort by the GLOBAL PBC Study group. Members of the
GLOBAL PBC group also aided with critical revision of the study for important intellectual
content and provided advice and suggestions.
This study was supported by unrestricted grants from Intercept Pharmaceuticals and was funded
by the Foundation for Liver and Gastrointestinal Research (a not-for-profit foundation) in
Rotterdam, the Netherlands and Toronto Centre for Liver Disease. The supporting parties had no
influence on the study design, data collection and analyses, or interpretation of the results.