# Soluble E-Cadherin: An Early Marker of Severity in Acute Pancreatitis

Authors: A. Sewpaul; J. J. French; T. K. Khoo; M. Kernohan; J. A. Kirby; R. M. Charnley
Journal: HPB Surgery
 (2009)

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## Abstract

Background/Aims. At present, there is no simple test for predicting severity in acute pancreatitis. We investigated the use of an assay of soluble E-cadherin (sE-cadherin). Methods. Concentrations of sE-cadherin, from 19 patients with mild acute pancreatitis, 7 patients with severe acute pancreatitis, 11 patients with other acute gastrointestinal pathologies, and 12 healthy subjects were measured using a commercially available sandwich ELISA kit based on two monoclonal antibodies specific to the extracellular fragment of human E-cadherin. Measurements were made at 12 hours or less from onset of pain and also at 24 and 48 hours after onset of pain. Results. Mean (standard deviation) concentration of sE-cadherin in patients with severe acute pancreatitis at <12 hours was 17780 ng/mL (7853), significantly higher than that of healthy volunteers 5180 ng/mL (1350), P=.0039, patients with other gastrointestinal pathologies 7358 ng/mL (6655), P=.0073, and also significantly higher than that of patients with mild pancreatitis, 7332 ng/mL (2843), P=.0019. Discussion. Serum sE-cadherin could be an early (within 12 hours) objective marker of severity in acute pancreatitis. This molecule warrants further investigation in the form of a large multicentre trial.


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## Body

## 1. Introduction

More than a century after
its comprehensive  description by Fitz [1],
acute  pancreatitis  remains a common disorder with  potentially devastating
consequences. The incidence has been reported to be as  high as 38 per 100 000
population per year [2]
with around 25% of patients developing severe or life-threatening complications
that require high-dependency nursing or intensive care support. Even though the
overall mortality over the past 30 years has fallen from 25%–30% to 6%–10%, it has
remained at that level for a decade or more [3].
If complications develop, however, mortality increases to 35% or higher [4],
with 50% of deaths occurring within one week of the attack, mostly from multiorgan
dysfunction syndrome (MODS) [5].It is generally
accepted that all patients with signs of moderate-to-severe acute pancreatitis should be
admitted to an intensive care unit and referred to specialised centres for
maximum supportive care [6–8]. As
complications may develop at any time, frequent reassessment and continuous
monitoring are necessary. The most important supportive therapy is adequate and
prompt fluid resuscitation with intravenous fluids and supplemental oxygen,
with a liberal indication for assisted or controlled ventilation to guarantee
optimal oxygen transport [7–9].To date, inhibition of
any known pathogenic step (by, e.g.,
octreotide, gabexate mesilate, and lexipafant) has not effectively reduced mortality
or increased long-term survival in severe acute pancreatitis [10–12]. Thus,
treatment of acute pancreatitis in the early stages is still symptomatic, with
no specific medication being currently available.UK guidelines [8]  recommend that severity stratification is attempted but
presently there is no tool that can predict, with a high degree of accuracy,
within 48 hours from the onset of pain, which patients will develop severe
disease. Initial clinical assessment alone
identifies fewer than half the patients with severe acute pancreatitis [13]. Scoring systems incorporating clinical, biochemical, or radiological
criteria for severity assessment have been in use for some decades. These
include the 11 criteria described by Ranson in 1970 [14], the
Glasgow score (eight criteria) [15], and the acute physiology and chronic health evaluation (APACHE II)
score (14 criteria) [16]. The sensitivity and specificity of these scoring systems for
predicting severe acute pancreatitis range between 55% and 90%, depending on
the cutoff number and the timing of scoring usually reaching optimal
sensitivity and specificity at 48 hours [17, 18]. The
ideal severity stratification system would be based on a simple-to-perform,
affordable, single, and quick test that is widely available and has a high
sensitivity and specificity very early in the disease process. The use of
single predictive markers has been investigated in the past.Serum amylase and
lipase, the standard tests for acute pancreatitis diagnosis, are poor
predictors of severity [19]. Novel markers for the early prediction of acute pancreatitis
severity include the pancreatic proenzyme trypsinogen-2 and its subunit TAP [20, 21], as
well as early inflammatory response markers such as serum IL-6 [22, 23], procalcitonin
[24, 25],
polymorphonuclear elastase [26], and
serum amyloid A [27]. The
more established marker C-reactive protein has been shown to be an accurate
severity predictor (sensitivity and specificity above 80%) at 48 hours postsymptom
onset if a cutoff level of 150 mg/L is used [6]. Unfortunately,
for a variety of reasons (poor predictive value before 48 hours, conflicting
reports, and difficult laboratory measurement), none of these single tests have
made the transition from the research setting into widespread clinical use.


### 1.1. E-Cadherin

E-cadherin is a 120-kDa transmembrane glycoprotein involved in the
calcium dependent adhesion of all epithelial cells.
Soluble E-cadherin is an 80-kDa peptide degradation product of the 120-kDa
E-cadherin molecule which is generated by a calcium ion dependent proteolytic
process [28, 29].
Matrix metalloproteinases, trypsin, kallikrein 7, and plasmin are examples of
molecules that are capable of performing this proteolytic process [28–37] (see Figure 1). The maintenance of
epithelial membranes in healthy individuals involves a continuous turnover of
E-cadherin, and independent of age and sex, low levels of sE-cadherin are found
in the serum [38].(a) The zipper
model for cadherin interaction showing homophilic interactions between
E-cadherin molecules expressed on the cell membranes; (b) proteolytic cleavage
of the extracellular portion of the E-cadherin fragment generating soluble
E-cadherin.
(a)(b)Elevated serum
levels of sE-cadherin are found in patients with certain malignancies,
including pancreatic
[30, 36, 38–45]. It
has been suggested that these increased levels are due to the cleavage of
membrane-bound E-cadherin by tumour-derived proteases.In a previous
study by Pittard et al., significantly elevated levels of sE-cadherin were found in patients exhibiting a
systemic inflammatory response. Importantly, these elevated sE-cadherin levels
were found within an early time frame of the disease process and correlated to
the severity of the developed inflammatory response [46].A systemic
inflammatory response is seen in acute pancreatitis. Therefore, it is probable
that sE-cadherin is cleaved from membrane-bound E-cadherin by inflammatory
proteases. If this also occurs early in the inflammatory response in acute
pancreatitis, and measurable serum levels of sE-cadherin relate to the severity
of the developing disease, then this molecule could be used to predict disease
severity. To test this hypothesis, we investigated, using a prospective pilot study,
whether serum levels of sE-cadherin measured at an early time point following
the onset of symptoms (within 12, 24, and 48 hours) in patients with acute
pancreatitis were significantly different in patients who went on to develop
mild, compared to severe disease. As serum levels of sE-cadherin are elevated
in many different inflammatory processes, its use in the diagnosis of acute
pancreatitis was not investigated as it is likely to be of limited value.


## 1.1. E-Cadherin

E-cadherin is a 120-kDa transmembrane glycoprotein involved in the
calcium dependent adhesion of all epithelial cells.
Soluble E-cadherin is an 80-kDa peptide degradation product of the 120-kDa
E-cadherin molecule which is generated by a calcium ion dependent proteolytic
process [28, 29].
Matrix metalloproteinases, trypsin, kallikrein 7, and plasmin are examples of
molecules that are capable of performing this proteolytic process [28–37] (see Figure 1). The maintenance of
epithelial membranes in healthy individuals involves a continuous turnover of
E-cadherin, and independent of age and sex, low levels of sE-cadherin are found
in the serum [38].(a) The zipper
model for cadherin interaction showing homophilic interactions between
E-cadherin molecules expressed on the cell membranes; (b) proteolytic cleavage
of the extracellular portion of the E-cadherin fragment generating soluble
E-cadherin.
(a)(b)Elevated serum
levels of sE-cadherin are found in patients with certain malignancies,
including pancreatic
[30, 36, 38–45]. It
has been suggested that these increased levels are due to the cleavage of
membrane-bound E-cadherin by tumour-derived proteases.In a previous
study by Pittard et al., significantly elevated levels of sE-cadherin were found in patients exhibiting a
systemic inflammatory response. Importantly, these elevated sE-cadherin levels
were found within an early time frame of the disease process and correlated to
the severity of the developed inflammatory response [46].A systemic
inflammatory response is seen in acute pancreatitis. Therefore, it is probable
that sE-cadherin is cleaved from membrane-bound E-cadherin by inflammatory
proteases. If this also occurs early in the inflammatory response in acute
pancreatitis, and measurable serum levels of sE-cadherin relate to the severity
of the developing disease, then this molecule could be used to predict disease
severity. To test this hypothesis, we investigated, using a prospective pilot study,
whether serum levels of sE-cadherin measured at an early time point following
the onset of symptoms (within 12, 24, and 48 hours) in patients with acute
pancreatitis were significantly different in patients who went on to develop
mild, compared to severe disease. As serum levels of sE-cadherin are elevated
in many different inflammatory processes, its use in the diagnosis of acute
pancreatitis was not investigated as it is likely to be of limited value.


## 2. Patients and Methods

### 2.1. Study Population

Patients admitted to
the Newcastle NHS hospitals with acute pancreatitis were assessed for inclusion
in the study. Acute
pancreatitis was defined as acute abdominal pain with a typical clinical
picture and a serum amylase level at least three times the upper limit of
normal and/or typical findings on computed tomography. We excluded patients who
had chronic pancreatitis, known malignancies, or were under 18 years. Clinical,
pathological, and radiological patient data was collected prospectively on a
database.We enrolled by parallel recruitment a number of patients admitted with
other abdominal inflammatory pathologies such as acute diverticulitis, perforated duodenal ulcer, cholangitis, acute appendicitis, and
acute cholecystitis. A third group of healthy volunteers was also recruited.
The same exclusion criteria applied to the patients with acute pancreatitis.


### 2.2. Study Design

Ethical approval was obtained from the Newcastle and North Tyneside
Health Authority, University of Newcastle upon Tyne and University of
Northumbria at Newcastle, and Joint Ethics Committee.Following informed
consent, three blood samples, for sE-cadherin measurement, were taken from each
individual. The first measurement was taken from the admission blood sample.
The subsequent three samples were taken at 12, 24, and 48 hours after the onset
of pain. Samples were subjected to centrifugation within 60 minutes and stored
at−20C°. sE-cadherin
levels were measured using a commercially available sandwich ELISA kit from
Takara Shuzo, Japan. In brief, the capture monoclonal antibody is coated onto
microtitre plate. Following blocking of nonspecific binding, patient samples,
or standard solutions is
incubated in the wells at 37C° for 2 hours. The detection
monoclonal antibody (conjugated with peroxidase) is then incubated in the above
wells at 37C° for 1 hour. The addition of peroxidase substrate
solution (H2O2 and tetramethylbenzibine) results in a
colour change. The reaction was terminated by the addition of 1 M H2SO4.
Absorbance was measured with the microtitre plate reader at 450 nm.
Each sample was measured three times and a mean value derived. Sample
concentrations were determined from standard curves obtained from standard solutions.Patients with local and/or organ failure were defined as having severe
acute pancreatitis according to the Atlanta classification [47].Data was analysed
using GraphPad PRISM (version 3.0). The data for sE-cadherin levels were
compared using the Mann-WhitneyU-test. Differences were considered as significant
when P<.05.


## 2.1. Study Population

Patients admitted to
the Newcastle NHS hospitals with acute pancreatitis were assessed for inclusion
in the study. Acute
pancreatitis was defined as acute abdominal pain with a typical clinical
picture and a serum amylase level at least three times the upper limit of
normal and/or typical findings on computed tomography. We excluded patients who
had chronic pancreatitis, known malignancies, or were under 18 years. Clinical,
pathological, and radiological patient data was collected prospectively on a
database.We enrolled by parallel recruitment a number of patients admitted with
other abdominal inflammatory pathologies such as acute diverticulitis, perforated duodenal ulcer, cholangitis, acute appendicitis, and
acute cholecystitis. A third group of healthy volunteers was also recruited.
The same exclusion criteria applied to the patients with acute pancreatitis.


## 2.2. Study Design

Ethical approval was obtained from the Newcastle and North Tyneside
Health Authority, University of Newcastle upon Tyne and University of
Northumbria at Newcastle, and Joint Ethics Committee.Following informed
consent, three blood samples, for sE-cadherin measurement, were taken from each
individual. The first measurement was taken from the admission blood sample.
The subsequent three samples were taken at 12, 24, and 48 hours after the onset
of pain. Samples were subjected to centrifugation within 60 minutes and stored
at−20C°. sE-cadherin
levels were measured using a commercially available sandwich ELISA kit from
Takara Shuzo, Japan. In brief, the capture monoclonal antibody is coated onto
microtitre plate. Following blocking of nonspecific binding, patient samples,
or standard solutions is
incubated in the wells at 37C° for 2 hours. The detection
monoclonal antibody (conjugated with peroxidase) is then incubated in the above
wells at 37C° for 1 hour. The addition of peroxidase substrate
solution (H2O2 and tetramethylbenzibine) results in a
colour change. The reaction was terminated by the addition of 1 M H2SO4.
Absorbance was measured with the microtitre plate reader at 450 nm.
Each sample was measured three times and a mean value derived. Sample
concentrations were determined from standard curves obtained from standard solutions.Patients with local and/or organ failure were defined as having severe
acute pancreatitis according to the Atlanta classification [47].Data was analysed
using GraphPad PRISM (version 3.0). The data for sE-cadherin levels were
compared using the Mann-WhitneyU-test. Differences were considered as significant
when P<.05.


## 3. Results

### 3.1. Patients

A total of 49
patients were recruited into the study; all met the eligibility criteria. 26
patients had acute pancreatitis, 12 had other acute abdominal pathologies, and
11 were healthy controls.The overall median
age of patients with acute pancreatitis was 56 (range 24–87); 12 were men.
19 patients had mild disease, and the remaining 7 had severe disease. The
aetiology was gallstones in 13 patients (9 mild and 4 severe). Alcohol was the
cause in 4 (3 mild and 1 severe). ERCP was the cause in 3 patients (2 mild and
1 severe). No obvious cause was found in 6 patients (5 mild and 1 severe). None
of the mild cases and 1 of the severe cases died.The median age of
the 12 healthy controls (4 male) was 29 (range 21–54).The median age of
the 11 patients with other acute abdominal pathologies (5 male) was 59 (range
29–72). The final
diagnoses for these patients were as follows: gastroenteritis 1; gastritis 1;
acute cholecystitis 2; acute appendicitis 2; diverticulitis 1; cholangitis 1;
perforated peptic ulcer 3.


### 3.2. Serum sE-Cadherin Levels

At less than 12 hours
from onset of pain, the mean (standard deviation) concentration of sE-cadherin
in patients with severe acute pancreatitis was 17780 ng/mL (7853),
significantly higher than that of healthy volunteers 5180 ng/mL (1350),P=.0039
(see Figure 2). In contrast, the
mean (SD) concentration of sE-cadherin in patients with mild acute pancreatitis
was 7332 ng/mL (2843), and the mean concentration of sE-cadherin in patients
with other pathologies was 7358 ng/mL (6655). The mean level in mild acute
pancreatitis patients was also significantly higher than that of healthy
volunteers (P=.0166), but not in those with other pathologies (P=.3909).
Importantly, significantly higher levels of sE-cadherin were detected in
patients with severe acute pancreatitis within 12 hours following onset of pain
(mean 17780 ng/mL) compared to mild pancreatitis (mean 7332 ng/mL), P=.0019
and those with other gastrointestinal pathologies (mean 7358 ng/mL), P=.0073.Figure 2
sE-cadherin concentration in
patients at 12 hours or less after onset of pain. Data are shown as a scatter
plot with the mean represented by a solid line.At 24 hours after
the onset of pain, the mean (SD) sE-cadherin concentration was 14320 ng/mL
(7532) in patients with severe acute pancreatitis, still significantly higher
than that of healthy volunteers 5518 ng/mL (1518),P=.0030 and also
compared to those with mild pancreatitis 6474 ng/mL (2823) P=.0065 (see Figure 3).Figure 3
sE-cadherin
concentration in patients at 24 hours after onset of pain. Data are shown as a
scatter plot with the mean represented by a solid line.At 48 hours the
mean (SD) sE-cadherin concentration was 13360 ng/mL (6440) in patients with
severe acute pancreatitis, still significantly higher than that of healthy
volunteers 4928 ng/mL (1314)P=.0080, and also compared to those with
mild pancreatitis 5818 ng/mL (1899) P=.0076
(see Figure 4).Figure 4
sE-cadherin
concentration in patients at 48 hours after onset of pain. Data are shown as a
scatter plot with the mean represented by a solid line.


## 3.1. Patients

A total of 49
patients were recruited into the study; all met the eligibility criteria. 26
patients had acute pancreatitis, 12 had other acute abdominal pathologies, and
11 were healthy controls.The overall median
age of patients with acute pancreatitis was 56 (range 24–87); 12 were men.
19 patients had mild disease, and the remaining 7 had severe disease. The
aetiology was gallstones in 13 patients (9 mild and 4 severe). Alcohol was the
cause in 4 (3 mild and 1 severe). ERCP was the cause in 3 patients (2 mild and
1 severe). No obvious cause was found in 6 patients (5 mild and 1 severe). None
of the mild cases and 1 of the severe cases died.The median age of
the 12 healthy controls (4 male) was 29 (range 21–54).The median age of
the 11 patients with other acute abdominal pathologies (5 male) was 59 (range
29–72). The final
diagnoses for these patients were as follows: gastroenteritis 1; gastritis 1;
acute cholecystitis 2; acute appendicitis 2; diverticulitis 1; cholangitis 1;
perforated peptic ulcer 3.


## 3.2. Serum sE-Cadherin Levels

At less than 12 hours
from onset of pain, the mean (standard deviation) concentration of sE-cadherin
in patients with severe acute pancreatitis was 17780 ng/mL (7853),
significantly higher than that of healthy volunteers 5180 ng/mL (1350),P=.0039
(see Figure 2). In contrast, the
mean (SD) concentration of sE-cadherin in patients with mild acute pancreatitis
was 7332 ng/mL (2843), and the mean concentration of sE-cadherin in patients
with other pathologies was 7358 ng/mL (6655). The mean level in mild acute
pancreatitis patients was also significantly higher than that of healthy
volunteers (P=.0166), but not in those with other pathologies (P=.3909).
Importantly, significantly higher levels of sE-cadherin were detected in
patients with severe acute pancreatitis within 12 hours following onset of pain
(mean 17780 ng/mL) compared to mild pancreatitis (mean 7332 ng/mL), P=.0019
and those with other gastrointestinal pathologies (mean 7358 ng/mL), P=.0073.Figure 2
sE-cadherin concentration in
patients at 12 hours or less after onset of pain. Data are shown as a scatter
plot with the mean represented by a solid line.At 24 hours after
the onset of pain, the mean (SD) sE-cadherin concentration was 14320 ng/mL
(7532) in patients with severe acute pancreatitis, still significantly higher
than that of healthy volunteers 5518 ng/mL (1518),P=.0030 and also
compared to those with mild pancreatitis 6474 ng/mL (2823) P=.0065 (see Figure 3).Figure 3
sE-cadherin
concentration in patients at 24 hours after onset of pain. Data are shown as a
scatter plot with the mean represented by a solid line.At 48 hours the
mean (SD) sE-cadherin concentration was 13360 ng/mL (6440) in patients with
severe acute pancreatitis, still significantly higher than that of healthy
volunteers 4928 ng/mL (1314)P=.0080, and also compared to those with
mild pancreatitis 5818 ng/mL (1899) P=.0076
(see Figure 4).Figure 4
sE-cadherin
concentration in patients at 48 hours after onset of pain. Data are shown as a
scatter plot with the mean represented by a solid line.


## 4. Discussion

In the clinical setting of a patient with acute pancreatitis,
initial therapy, accurate severity stratification, and an appropriate facility
for patient management are of major interest for the admitting clinician. At
present, there is no method by which to accurately predict severity. Many
scoring systems have been proposed but all have their drawbacks. The
possibility that an affordable, quick, single, and accurate test may exist has
led clinicians to investigate numerous (mainly inflammatory mediators)
biochemical molecules. Many have been assessed and detected either in serum or
urine but for a number of reasons have failed to reach the clinical setting.This study has
shown that serum levels of sE-cadherin can be used to predict severity of acute
pancreatitis at an early time point, with the mean differences in sE-cadherin
concentration being statistically different within 12 hours or less or at 24
hours after the onset of pain. Furthermore, although not presently widely available,
this test is quick, affordable (comparable to CRP), and could easily be
incorporated into hospital practice. It is thought
that sE-cadherin, elevated  levels of which are found in inflammatory
conditions, originates
via cleavage from membrane-bound E-cadherin. The mechanism by which and at exactly which point during
an inflammatory process this occurs is not presently fully understood. Experimentally, molecules
such as metalloproteinases, kallikrein 7, plasmin,  and trypsin have all been
shown to be capable of performing this cleavage process
[28–37]. With
regard to metalloproteinases, it is known that they can cause organ damage [48] and
have a tissue remodelling role in the regeneration following an attack of
pancreatitis, but recently evidence is emerging that they could be important in
early inflammatory events.  Not only have
elevated levels of MMP-2 and MMP-9 been reported in the peritoneal fluid of
rats following sodium taurocholate-induced pancreatitis [48], but
also MMP-3 and MMP-9 enzyme activity has been shown to be elevated 12 hours after
cerulein-induced pancreatitis [49].
Therefore, trypsin and/or MMP activation very early in the pathogenesis of
acute pancreatitis could be one mechanism by which the elevated levels of
sE-cadherin are achieved.In a recent study,
Steinhusen et al. [35] reported that during apoptosis,
fragments of E-cadherin with apparent molecular masses of 24, 29, and 84 kDa
were generated by two distinct proteolytic activities. In addition to a
caspase-3-mediated cleavage releasing the cytoplasmic domain of E-cadherin, a
metalloproteinase sheds
the extracellular domain from the cell surface during apoptosis. In experimental pancreatitis, it has been found
that acute pancreatitis is associated with the induction of acinar cell
apoptosis, the degree of which mimics the severity of pancreatitis [50].The mean sE-cadherin concentrations were higher in our healthy
controls compared with those reported in one study (5180 ng/mL versus 2515 ng/mL,
resp.) [40], but similar to levels found in a more recent
study (5616 ng/mL) [42]. Racial differences between groups have been
suggested as an explanation, but we suspect that the ELISA substrate used is
the reason. The substrate used was o-Phenylenediamine (OPD) in the former study [40] and 3,3′,5,5′-Tetramethylbenzidine
(TMB) in our own  and
the latter study [42] where comparable sE-cadherin levels were found.Prior to our
study, we suspected that raised sE-cadherin levels would be found in all
patients experiencing an inflammatory event. From our results, it appeared that
this was not the case, and that raised sE-cadherin levels were only found in
patients with an inflammatory insult that resulted in significant multiorgan
failure. The solitary high sE-cadherin level measured in the group of patients
with other pathologies was from a patient with cholangitis who did go on to
develop multiorgan failure.Of the seven
patients who had the severe form of acute pancreatitis, 6 had significantly
elevated levels of sE-cadherin within 12 hours from the onset of pain. These
patients required admission to critical care for organ support within 24 hours
of their presentation to hospital and went on to develop MOF. The remaining
patient, whose sE-cadherin level was not significantly raised within 12 hours after
the onset of pain, is worthy of further discussion. This female patient
presented with gallstone pancreatitis was managed initially on the general
ward. A further bout of pain, however, developed on day 4 following admission
for which she needed HDU care on day 5. She went on to develop multiorgan failure
and pancreatic necrosis. It is possible that there  may have been a second
attack of pancreatitis induced on day 4 in an inpatient and that she subsequently
developed severe acute pancreatitis and necrosis from this second attack.
Unfortunately, we do not have serum sE-cadherin levels relating to this second
bout of pain.Despite the small sample size in this study, sE-cadherin seems to be
an exciting potential very early marker (within 12 hours of onset of symptoms)
of severity in acute pancreatitis. It is noted however that this is a small pilot study and
that firm conclusions cannot be drawn until the hypothesis has been tested on a
larger population. A larger sample
size would permit more sophisticated statistical analysis. The predictive value of sE-cadherin could then
be evaluated by receiver-operating characteristic (ROC) curves to determine the
optimal cutoff value to predict severe AP.  Sensitivity and specificity, as well as positive
and negative predictive values, could then be calculated. This would also allow
for a useful comparison of the prognostic value of E-cadherin with other
predictive scores. We therefore feel
that this molecule
warrants further study in the form of a multicentre trial.



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*Source: 397375-2009-04-27.xml*