articulo

1. Biomarkers to estimate the probability of complicated appendicitis☆
Meghan C. Daly a,b,
⁎, Daniel von Allmen c
, Hector R. Wong a,d
a
Division of Critical Care Medicine, Cincinnati Children's Hospital Medical Center and Cincinnati Children's Research Foundation, Cincinnati, OH, USA
b
Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, OH, USA
c
Department of Surgery, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA
d
Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 10 January 2017
Received in revised form 2 August 2017
Accepted 2 September 2017
Key words:
Appendicitis
Biomarker
Matrix metalloproteinases
Tissue inhibitors of metalloproteinase
Conservative
Background: The conventional paradigm that all children with appendicitis require an appendectomy is being
challenged by the idea that some patients may be successfully managed non-operatively. The study aimed to de-
termine if matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) are candidate
biomarkers for estimating the probability of complicated appendicitis in pediatric patients.
Methods: The study was a single-institution, prospective cohort study. MMP and TIMP serum protein concentra-
tions were measured in patients with suspected appendicitis. Three hundred and thirty-one patients were en-
rolled with appendicitis. Classification and Regression Tree (CART) analysis was used to determine the
combination of candidate biomarkers that best predicted complicated appendicitis.
Results: The CART-generated decision tree for the derivation cohort included WBC count, MMP-8, MMP-9, MMP-
12, TIMP-2, and TIMP-4 and had the following test characteristics for estimating the probability of complicated
appendicitis (95% CI): AUC 0.86 (0.81–0.90); sensitivity 91% (83–96); specificity 61% (53–68); positive predictive
value 58% (50–66); negative predictive value 92% (84–96); positive likelihood ratio (LR) 2.3 (1.9–2.8); and neg-
ative LR 0.15 (0.08–0.3).
Conclusions: MMPs and TIMPs have the potential to serve as biomarkers to estimate the probability of complicat-
ed appendicitis in pediatric patients. The multi-biomarker-based decision tree has test characteristics suggesting
clinical utility for decision making.
Level of Evidence: Level II: Study of Diagnostic Test.
Published by Elsevier Inc.
Acute appendicitis is the most common condition requiring urgent
abdominal surgery in children [1]. The longstanding rationale for ap-
pendectomy is the prevention of complications secondary to disease
progression and perforation [2]. However, the traditional paradigm
that all children with appendicitis require appendectomy is now being
challenged by the concept that a subgroup of children with low risk of
disease progression can be successfully managed non-operatively with
antibiotics and supportive care alone [3–10]. A pilot study was per-
formed to investigate the feasibility of this non-operative management
approach [11]. A recent meta-analysis reported success rates of non-
operative management to be 91% at 30-day follow-up and 73% at one-
year follow-up [12]. Another prospective observational study using
rigorous inclusion criteria demonstrated success rates of non-
operative management to be 87% at 18-month follow-up [13].
Given the increasing interest in the non-operative management of
appendicitis, it is crucial to reliably identify patients appropriate for
non-operative management. A companion diagnostic test to reliably es-
timate which patients can be safely managed in this manner would be
highly valuable. Our preclinical studies have identified matrix
metalloproteinase-8 (MMP8) as an important mediator of intestinal in-
jury in animal models [14,15]. We hypothesized that MMP-8, other ma-
trix metalloproteinase family members, and endogenous tissue
inhibitors of metalloproteinases (TIMPs) can serve as companion diag-
nostic biomarkers to estimate the probability of complicated appendici-
tis in pediatric patients.
1. Methods
1.1. Patient cohort and data collection
This is a single institution, prospective cohort study. The study
conformed to good clinical practice guidelines and followed the recom-
mendations of the Declaration of Helsinki. The study protocol was ap-
proved in September 2014 by the Cincinnati Children's Hospital
Journal of Pediatric Surgery 53 (2018) 437–440
☆ Conflicts of Interest and Source of Funding: The authors have no competing interests
to report. The study was supported by NIH T32GM008478, R01GM099773, and
R01GM108025.
⁎ Corresponding author at: University of Cincinnati Medical Center, 231 Albert Sabin
Way, ML 0558, Cincinnati, OH 45267. Tel.: +1 315 569 2351; fax: +1 513 558 3474.
E-mail address: megcnolan@gmail.com (M.C. Daly).
https://doi.org/10.1016/j.jpedsurg.2017.09.004
0022-3468/Published by Elsevier Inc.
Contents lists available at ScienceDirect
Journal of Pediatric Surgery
journal homepage: www.elsevier.com/locate/jpedsurg

2. Medical Center (CCHMC) Institutional Review Board (ID 2014–5392).
The CCHMC IRB granted a waiver of informed consent.
Pediatric patients presenting to the emergency department with
suspected appendicitis, and a serum sample obtained at the time of pre-
sentation were eligible for study enrollment. The primary outcome vari-
able was complicated appendicitis as defined by an abscessed,
perforated, or gangrenous appendix at the time of appendectomy. Both
the operative report and pathology report were reviewed to determine
a classification of complicated appendicitis. Study subjects were followed
to determine their clinical course after evaluation for appendicitis.
1.2. Candidate biomarkers
MMP and TIMP serum protein concentrations were measured using
a multiplex magnetic bead-based immunoassay designed by BIORAD
Corporation (Hercules, CA, USA) and a Luminex 100/200 System
(Luminex Corporation, Austin, TX, USA), according to the manufactures'
specifications. The multiplex panel included measurements for MMP-1,
-2, -3, -7, -8, -9, -10, -12, -13 and TIMP-1, -2, -3, -4.
1.3. Statistical analysis
The primary outcome variable for all modeling procedures was the
presence of complicated appendicitis. Descriptive statistics and compar-
isons were conducted using SigmaStat Software (Systat Software, Inc.,
San Jose, CA, USA). Receiver operating characteristic curves were con-
structed to determine the ability of each candidate biomarker to esti-
mate the probability of complicated appendicitis. Classification and
Regression Tree (CART) methodology (Salford Predictive Modeler
v8.0, Salford Systems, San Diego, CA) was used to determine the combi-
nation of candidate biomarkers that best predicted complicated appen-
dicitis [16]. All 13 candidate biomarkers, as well as white blood cell
(WBC) count were considered as predictor variables in the CART analy-
sis. Weighting of cases or introducing a cost for incorrect predictions
was not used in the modeling procedures. The derivation cohort was
randomly selected to include 80% of the study cohort; the test cohort
consisted of the remaining 20%. Performance of the derivation and test
cohort decision trees are reported using diagnostic test statistics with
95% confidence intervals. Statistical significance was defined as p b 0.05.
2. Results
2.1. Derivation of the decision tree
Five hundred and eighty-seven subjects who presented to the emer-
gency department with suspected appendicitis were enrolled in the
study. Among these, 331 had surgically confirmed appendicitis, and
125 (38%) of these had complicated appendicitis. The primary analysis
is based on these 331 subjects with appendicitis. We randomly selected
80% of these subjects (n = 265) for the derivation cohort. The demo-
graphic and clinical characteristics of the study subjects in the deriva-
tion cohort are depicted in Table 1. Among the individual candidate
biomarkers, MMP-8 had the greatest area under the curve (AUC =
0.68; p b 0.001) for estimating the probability of complicated appendi-
citis. MMP-9, MMP-10, MMP-12, MMP-13, TIMP-2 and TIMP-4 also
had AUCs approaching 0.60 and p values ≤0.05 (Table 2). WBC count
alone had an AUC of 0.77, p b 0.001.
Fig. 1 shows the derived decision tree. Maximum accuracy for esti-
mating the risk of complicated appendicitis was attained with 5 of the
13 candidate biomarkers: MMP-8, −9, −12, TIMP2, and TIMP4. There
were three low-risk terminal nodes (≤ 20.8% risk of complicated appen-
dicitis; nodes 1, 3, and 7), two moderate risk terminal nodes
(27.5–47.6% risk of complicated appendicitis; nodes 2 and 4), and
three high-risk terminal nodes (59.1–90.0% risk of complicated appen-
dicitis; nodes 5, 6, and 8). Table 3 shows the diagnostic test characteris-
tics of the decision tree for the derivation cohort, wherein subjects in the
low-risk terminal nodes are classified as predicted to not have compli-
cated appendicitis, and subjects in the moderate and high risk terminal
nodes are classified as predicted to have complicated appendicitis.
2.2. Testing the decision tree
The demographic and clinical characteristics of the study subjects in
the test cohort (n = 66) are depicted in Table 1. The test cohort subjects
were classified based on the derived decision tree, without any modifi-
cations. Table 4 shows the diagnostic test characteristics of the decision
tree in the test cohort.
2.3. Secondary considerations
Fig. 2 compares the receiver operating characteristic (ROC) curves
for the decision tree and WBC count alone for all subjects in the deriva-
tion and test cohorts. The AUC of the decision tree (0.86; 95% CI: 0.82 to
0.90) was superior to that of WBC count alone (0.77; 95% CI: 0.72 to
0.82; p = 0.0003) for estimating the risk of complicated appendicitis.
Ten of the initial 587subjects enrolled were taken to the operating
room for an appendectomy but were found to have a normal appendix.
All of these patients fell into low-risk terminal nodes (nodes 1, 3, or 7).
The decision tree would have identified these subjects as having a low
probability of complicated appendicitis and they might have avoided
an operation under a non-operative management protocol.
Table 1
Demographic and clinical characteristics of the derivation and test cohorts.
Derivation cohort (n = 265) Test cohort (n = 66)
Uncomplicated Complicated Uncomplicated Complicated
N (% total) 165 (62) 100 (38) 41 (62) 25 (38)
# Males (%) 104 (63) 65 (65) 21 (51) 13 (52)
# Females (%) 61 (37) 35 (35) 20 (49) 12 (48)
Median age
years (range)
11 10 10 10
[IQR] [9–14] [7–12.5] [8–13] [8–12]
# for race (%)
Caucasian 129 (78) 82 (82) 35 (85) 19 (76)
African American 17 (10) 12 (12) 2 (5) 2 (8)
Other 19 (12) 6 (6) 4 (10) 4 (16)
# with temp
N38 °C (%)
8 (5) 37 (37) 6 (15) 9 (36)
# with
vomiting (%)
85 (52) 69 (69) 20 (49) 15 (60)
Table 2
Areas under the receiver operating curves for individual biomarkers.
Biomarker AUC 95% CI p value
Higher value associated with complicated appendicitis
MMP1 0.51 0.50–0.58 0.667
MMP2 0.52 0.46–0.59 0.502
MMP3 0.56 0.50–0.62 0.071
MMP7 0.51 0.50–0.58 0.658
MMP8 0.68 0.63–0.74 b0.001
MMP9 0.62 0.56–0.69 b0.001
MMP10 0.59 0.53–0.65 0.006
MMP13 0.57 0.51–0.63 0.031
TIMP4 0.58 0.52–0.64 0.012
WBC 0.77 0.71–0.84 b0.001
Lower value associated with complicated appendicitis
MMP12 0.57 0.51–0.64 0.025
TIMP1 0.54 0.48–0.60 0.223
TIMP2 0.61 0.55–0.67 b0.001
TIMP3 0.52 0.45–0.58 0.618
438 M.C. Daly et al. / Journal of Pediatric Surgery 53 (2018) 437–440

3. 3. Discussion
We have derived a biomarker-based risk stratification tool to reliably
estimate the risk of complicated appendicitis in pediatric patients. Reli-
ably estimating this risk is a key decision point for determining which pa-
tients are potentially eligible for non-operative management of acute
appendicitis, and which patients warrant traditional appendectomy. The
ideal characteristics of a companion test that informs clinical decision
making regarding non-operative management of appendicitis include a
high sensitivity, a high negative predictive value, and a low negative like-
lihood ratio. The decision tree demonstrated these test characteristics in
both the derivation and test cohorts. Such test characteristics would
allow for reliable inclusion of low-risk patients in a non-operative man-
agement protocol, with simultaneous exclusion of higher risk patients.
The potential advantages of successful non-operative management of
appendicitis, without the need for surgery, are self-evident. Peri-operative
complications occur in approximately eight to 11% of patients undergoing
appendectomy and recovery may require absence of up to one week of
school [17,18]. Several recent trials of non-operative management of ap-
pendicitis have reported no increased risk of developing perforated ap-
pendicitis [8,11,19,20]. Initial cost-effective analysis supports the use of
non-operative management in uncomplicated appendicitis [7,21]. How-
ever, non-operative management has been associated with more subse-
quent emergency room visits and hospitalizations compared to those
patients managed operatively [22]. The impact of medical interventions
on patient-centered outcomes is becoming increasingly important in
the current era of demand-side strategic purchasing.
In our cohort, individual biomarkers demonstrated significantly
lower AUCs than the derived decision tree, with MMP-8 having the
greatest AUC of 0.68. WBC has been traditionally utilized as a supportive
laboratory finding in the diagnosis of appendicitis [23]. Sensitivity and
specificity of WBC count in this setting have been reported to range
from 70% to 80% and 60% to 68%, respectively [24–27]. The AUC of the
WBC count alone in our cohort was 0.77 for estimating the risk of com-
plicated appendicitis. This was inferior to that of the decision tree, indicat-
ing that the decision tree provides predictive information beyond that of
Fig. 1. The classification tree. The classification tree includes matrix metalloproteinase-8 (MMP-8), MMP-9, MMP-12, WBC count, tissue inhibitor of metalloproteinase 2 (TIMP2), and TIMP4. The
MMP and TIMP data are shown as ng/ml. The root node provides the total number of samples, and the number of samples associated with no disease activity and disease activity, with the
respective rates. Each daughter node provides the respective decision rule criterion and the number of samples associated with no disease activity and disease activity, with the respective
rates. Terminal nodes (TN) TN1, TN3, and TN7 are low-risk terminal nodes (≤20.8% risk of complicated appendicitis). TN2 and TN4 are moderate risk terminal nodes (27.5 to 47.6% risk of
complicated appendicitis) and TN5, TN6, and TN8 are high-risk terminal nodes (59.1 to 90.0% risk of complicated appendicitis). To calculate the diagnostic test characteristics, all low-risk
samples are predicted to be associated with uncomplicated appendicitis, while all intermediate and high-risk samples are predicted to be associated with complicated appendicitis.
Table 3
Diagnostic test characteristics of the classification tree for estimating the risk of complicat-
ed appendicitis in the derivation cohort.
Category
# of true positives 91
# of false positives 65
# of true negatives 100
# of false negatives 9
Sensitivity 91% (83–96)
Specificity 61% (53–68)
Positive predictive value 58% (50–66)
Negative predictive value 92% (84–96)
Positive likelihood ratio 2.3 (1.9–2.8)
Negative likelihood ratio 0.15 (0.08–0.3)
Area under the curve 0.86 (0.81–0.90)
Table 4
Diagnostic test characteristics of the classification tree for estimating the risk of complicat-
ed appendicitis in the test cohort.
Category
# of true positives 24
# of false positives 14
# of true negatives 27
# of false negatives 1
Sensitivity 96% (78–100)
Specificity 66% (49–79)
Positive predictive value 63% (46–78)
Negative predictive value 96% (80–100)
Positive likelihood ratio 2.8 (1.8–4.3)
Negative likelihood ratio 0.06 (0.01–0.4)
Area under the curve 0.85 (0.76–0.94)
439
M.C. Daly et al. / Journal of Pediatric Surgery 53 (2018) 437–440

4. the WBC count alone. Given the shortcomings of a single biomarker to ac-
curately detect the presence of complicated appendicitis in pediatric pa-
tients, a combination of biomarkers may be more advantageous. To our
knowledge, we are not aware of a validated stratification tool assessing
the probability of complicated appendicitis that performs in an equivalent
manner to that of our derived classification tree.
We note the limitations of our study. CART analysis has the potential
to over-fit a dataset. We attempted to address this issue by dividing our
cohort into separate derivation and test cohorts. Nonetheless, our model
requires further validation in a prospectively enrolled cohort. In addi-
tion, the panel of biomarkers used to estimate the risk of complicated
appendicitis is currently available only as research-based assays and
they require batching. In order to use these biomarkers for clinical deci-
sion making, a rapid assay would need to be developed. The technology
to develop such a test currently exists.
To conclude, these data suggest that MMPs and TIMPs have the potential
to serve as biomarkers to estimate the probability of complicated appendici-
tis in pediatric patients. The combination of MMP-8, MMP-9, MMP-12,
TIMP-2, and TIMP-4 produced the most reliable test characteristics. The
multi-biomarker-based decision tree has test characteristics suggesting clin-
ical utility for decision making and may serve to identify pediatric patients
with appendicitis that can be safely managed non-operatively.
Acknowledgments
The authors would like to thank Patrick Lahni for the technical support.
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Fig. 2. Comparison of the receiver operating characteristics (ROC) curves for the classification tree and WBC count in all subjects. The ROC for the classification tree is shown as a black line,
and the ROC for WBC count is shown as a gray line. The area under the curve for the classification tree (0.86, 95% CI: 0.82 to 0.90) was greater than that of the WBC count alone (0.77, 95% CI:
0.72 to 0.82, p = 0.0003).
440 M.C. Daly et al. / Journal of Pediatric Surgery 53 (2018) 437–440