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A machine learning approach identified a diagnostic model for pancreatic

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DOI: 10.1016/j.pan.2020.07.399

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Contents lists available at ScienceDirect

Pancreatology
journal homepage: www.elsevier.com/locate/pan

A machine learning approach identified a diagnostic model for

pancreatic cancer through using circulating microRNA signatures
Behrouz Alizadeh Savareh a, d, Hamid Asadzadeh Aghdaie b, Ali Behmanesh c, **,
Azadeh Bashiri d, Amir Sadeghi b, Mohammadreza Zali b, Roshanak Shams b, e, *
a
PhD in Medical Informatics, National Agency for Strategic Research in Medical Education, Tehran, Iran
b
Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
c
Student Research Committee, School of Health Management and Information Sciences, Iran University of Medical Sciences, Tehran, Iran
d
Department of health information management, school of management and medical information sciences, Shiraz University of Medical Sciences, Shiraz,
Iran
e
Department of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Late diagnosis of pancreatic cancer (PC) due to the limited effectiveness of modern testing approaches,
Received 21 January 2020 causes many patients to miss the chance of surgery and consequently leads to a high mortality rate.
Received in revised form Pivotal improvements in circulating microRNA expression levels in PC patients make it possible to di-
29 June 2020
agnose and treat patients at earlier stages. A list of circulating miRNAs was identified in this study using
Accepted 25 July 2020
Available online 9 August 2020
bioinformatics methods in association with pancreatic cancer through analyzing four GEO microarray
datasets. The value of top miRNAs was then assessed via using a machine learning method. Taking the
advantage of a combinatorial approach consisting of Particle Swarm Optimization (PSO) þ Artificial
Keywords:
Micro RNA
Neural Network (ANN) and Neighborhood Component Analysis (NCA) iterations on a collection of top
Circulating miRNA differentially expressed circulating miRNAs in PC patients, facilitated ranking them by significance.
Pancreatic cancer MiRNA’s functional analysis in the final index was performed by predicting target genes and constructing
Bioinformatics PPI networks. Remarkably, the final model consist of miR-663a, miR-1469, miR-92a-2-5p, miR-125b-1-3p
Early detection and miR-532e5p showed great diagnostic results on investigated cases and the validation set (Accuracy:
Machine learning 0.93, Sensitivity: 0.93, and Specificity: 0.92). Kaplan-Meier survival assessments of the top-ranked
miRNAs revealed that three miRNAs, hsa-miR-1469, hsa-miR-663a and hsa-miR-532e5p, had mean-
ingful associations with the prognosis of patients with pancreatic cancer. This miRNA index may serve as
a non-invasive and potential PC diagnostic model, although experimental testing is needed.
© 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Introduction challenging [2]. The average survival time following diagnosis is

Pancreatic ductal adenocarcinoma (PDAC) is one of the most
mortal malignancies and a big health problem worldwide [1].
Resection surgery can raise the 5-year survival rate to 20%, but
only 10e20% of the patients find a chance for this treatment since
early detection at a treatable stage of the disease is mainly
* Corresponding author. Gastroenterology and Liver Diseases Research Center,
Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti Uni-
versity of Medical Sciences, Tehran, Iran.
** Corresponding author.
E-mail addresses: aa.behmanesh@gmail.com (A. Behmanesh), Shams.rosha.86@
gmail.com (R. Shams).
often fewer than 6 months [3]. Late diagnosis of pancreatic
cancer (PC) due to the limited efficacy of conventional diagnostic
methods can cause many patients to miss the chance of treat-
ment by surgery and, lead to a high mortality rate [4]. Numerous
molecular components such as CA19-9 and CA125 have been
introduced as potential markers for detection of pancreatic tu-
mors [5,6] however they are not sufficiently sensitive and specific
to significantly discriminate cancer from healthy or benign con-
ditions [7]. MicroRNAs (miRNAs) have been represented as
promising biomarkers for diagnosis of PC in recent decades [8].
These are comprising a group of short non-coding RNA molecules
with 19e25 nucleotides that have been considered as hopeful
biomarkers of early cancer diagnosis and precise prognosis [9].

https://doi.org/10.1016/j.pan.2020.07.399
1424-3903/© 2020 IAP and EPC. Published by Elsevier B.V. All rights reserved.
1196 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
Some miRNAs play crucial roles in the progression of cancer and
aberrant expression of those miRNAs may cause the development
of many types of tumors [10,11]. That means identifying proper
miRNAs will be helpful in the diagnosis of the disease [12].
Computational approaches, specifically machine learning, have
been used by many scientists for cancer diagnosis and classifi-
cation through using big datasets consist of miRNAs as bio-
markers [13e16]. In this study, we identified a number of
significantly differential serum miRNAs expressed in 671 micro-
array PDAC expression profiles using bioinformatics techniques.
The purpose of the study was to develop a diagnostic model for
PDAC using a combination of bioinformatics methods plus ma-
chine learning approaches -including Particle Swarm Optimiza-
tion (PSO), Artificial Neural Network (ANN) and Neighborhood
Component Analysis (NCA). Accordingly, it was critical to confine
the large number of miRNAs associated with PDAC to detect the
most discriminative of miRNA features subsets pursuing fulfill
distinct purposes. Then these miRNAs are accounted directly as
features on which feature selection methods can be applied to
remove remaining irrelevant and unnecessary ones. In machine
learning, feature selection is a technique of selecting a subset
from a given set of features based on certain criteria without
alteration of the original features, which retains the interpreta-
tion of the results. This hinders overfitting and boosts classifi-
cation performance specifically with gene expression data, which
usually has a high number of features. Therefore we focused on
those top significant miRNA signatures that had the lowest
adjusted p-values and highest Fold Change logarithm (log-FC) in
the results of our bioinformatics analyses. We then performed
feature selection methods on this diminished group of miRNAs to
find out which ones are more critical for cancer diagnosis.
Although some of those miRNAs have already been experimen-
tally proved to be associated with pancreatic cancer, through
machine learning technique assistance, we attempted to
demonstrate the association which can serve as a diagnostic
model for PDAC.
Material & methods
Microarray datasets selection and identification of differentially
expressed miRNAs
In order to find the most promising diagnostic miRNA bio-
markers for PDAC, a total of four GEO profiles (GSE113486,
GSE59856, GSE85589, and GSE106817) consisting of 671 selected
miRNA expression profiles from serum samples of PDAC patients
and healthy controls were downloaded and considered for differ-
ential expression analysis. By the use of a visual analytics platform
for comprehensive gene expression profiling & meta-analysis on-
line tool (NetworkAnalytes, V3.0) [17], merging and analysis of the
data of all 671 PDAC patients and Differential expression analysis
were done. NetworkAnalyst 3.0 is a powerful online data analytics
platform which is specialized in gene expression data tran-
scriptome profiling, network analysis and meta-analysis. Inte-
grating statistical significance (p values) with biological context
(fold changes) from different sources of datasets, allows users to
easily identify the most promising gene candidates from differen-
tial expression analysis results. All data were uploaded, normalized
with Log 2 transformation and DE analysis for Cancer vs Control
with P: 0.05 was performed. At the next step, the integrity of the
data was checked. For meta-analysis, Fisher’s method with P: 0.05
cut off was applied to combine the p-values. Top miRNAs with
lowest p-values and logarithm of fold change (log-FC) ±2 were
selected for further evaluations.
Machine learning
In order to identify miRNA signatures that are the most prom-
ising markers in the diagnosis of PDAC, a procedure was designed
using a combination of data mining techniques, illustrated in Fig. 1.
Each of which are briefly described.
Normalization
Normalization technique is applied for data preparation in
machine learning only when features have different ranges.
Normalization has a positive effect on the accuracy of the models.
This technique changes the values of variables to a common scale,
without losing the useful information. Min-Max Normalization is
one of the common methods which normalizes variables between
0 and 1, as the following formula:
X org  Min
X norm ¼ (1)
Max  Min
Feature selection
In this section, the most useful features in a dataset were
selected. The goal of features selection is decreasing training time,
as well as increasing model interpretability, and generalization
performance on the test set. To select the most useful features in a
dataset, two separate paths of techniques were used: 1) combina-
tion of Particle Swarm Optimization (PSO) and Artificial Neural
Network (ANN) to select features, 2) applying Neighborhood
Components Analysis (NCA) in feature selection from another
perspective. Each of the mentioned methods described in more
details. Moreover the reasons for choosing each of the methods will
be stated.
Particle Swarm Optimization (PSO). As the number of features
grows, choosing the more important ones with lowest classification
error requires exponential order of computations. Because of the
space problem of 28 dimensions (features representing gene
expression), selecting the most optimal subset requires high cal-
culations. Therefore, an appropriate approach to selecting features
in such a situation can be determined by heuristic methods such as
PSO. The use of PSO due to the relatively high speed in optimization
as well as the stability in finding the optimal answers, is an
attributed approach and is fully compatible with the requirements
of this study. Therefore, PSO technique was used to optimize this
study. In the PSO, the search can be carried out by the speed of the
particle. During the development of several generations, only the
most optimist particle can transmit information onto the other
particles, hence the speed of the researching is very fast. Conse-
quently, the calculation in PSO is very simple. Compared with the
other developing calculations, it can be completed easily [18]. Every
particle in the population has two vectors, i.e., velocity vector and
position vector [19]. The PSO algorithm is recursive. It prompts
social search behavior among particles in the search space, where
every particle indicates one point in n-dimensional space as a so-
lution for a given problem. In comparison with other EAs such as
Genetic Algorithm (GA), the PSO has improved search efficacy with
faster and more stable convergence rates [20]. The approach for
PSO used in this study is based on the standard PSO [21], but ac-
cording to the situation, the following changes were applied.
Feature selection is encoded in the binary format, and in the
evolutionary iterations, the PSO tries to find a binary code that
minimizes the classification error in PC classification. Then, features
that have a stronger correlation with PC classification are identified
through the PSO iterations. The PSO select more important features
 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
that are fed to ANN (for fitness evaluation).
Artificial neural network (ANN). Due to the simulation of the human
brain’s decision-making process in the form of mathematical
models, the artificial neural network has been successful in
analyzing nonlinear relationships and is therefore superior to other
machine learning techniques.
Based on the nonlinear and complex relationships between the
input variables (circulating microRNA signatures) and the predicted
result (pancreatic cancer), ANN was considered as the fitness
function in the PSO mechanism. Also ANN being fast, accurate and
reliable in the prediction or approximation, especially when nu-
merical and mathematical methods fail. Moreover there is a sig-
nificant simplicity in using ANN due to its power to deal with
multivariate and complicated problems(22).
An artificial neural network, a supervised learning algorithm,
includes an interconnected group of artificial neurons in the form of
intermittent layers. It processes information through a con-
nectionist method to computation. At first, to calculate the error,
the ANN randomly makes predictions which are compared with the
target outputs. ANN objective is to minimize the error. The ANN,
adaptive system, changes its structure (weight among neurons)
during a training phase to minimize the error [23]. Fig. 2 demon-
strates the general structure of an ANN. Based on the generic
structure; ANN designed to predict Pancreatic Cancer was as fol-
lows: the input layer contains circulating microRNA signatures,
whereas a hidden layer has been used, also the output layer con-
tains the PC classification. The hidden layer was constructed with
15 neurons (three times the number of inputs, according to the rule
of thumb). The activation function in the hidden layer was
considered as ‘tansig’, also ‘softmax’ selected for the output layer.
These activation functions are appropriate for pattern recognition
ANN’s [24]. In order to implement PC predictor model in Matlab
2019, pattern recognition networks (pattern-net) are used for
training and testing ANN. The pattern nets are feed-forward net-
works that can be trained to classify observation cases according to
target.
Neighborhood Component Analysis (NCA). In addition to PSO and
ANN combination, the other mechanism used to select important
features was based on the application of the Neighborhood
Component Analysis. Neighborhood Component Analysis (NCA) is
a non-parametric method for selecting features with the goal of
maximizing prediction accuracy of regression and classification
algorithms. The reason to use NCA is that the nearest neighbor,
NCA’s core, is a simple and efficient nonlinear decision rule and
often yields competitive results compared with the state-of-the-
art classification methods [25]. Nearest neighbor (KNN) is
extremely simple yet surprisingly effective method for classifica-
tion. Its appeal stems from the facts that its decision surfaces are
nonlinear, there is only a single integer parameter (which is easily
tuned with cross-validation), and the expected quality of pre-
dictions improves automatically as the amount of training data
increases [26].
NCA has some advantages over other methods like PCA, LDA, etc.
With low dimensionality projection, the classes are consistently
much better separated by the NCA transformation than by either
PCA (which is unsupervised) or LDA (which has access to the class
labels). Of course, the NCA transformation is still only a linear
projection, just optimized with a cost function which explicitly
encourages local separation. Furthermore, we can apply a nearest-
neighbor classification in the projected space. Using the same
projection learned at training time, it projects the training set and
all future test points and performs KNN in the low-dimensional
space using the Euclidean measure [26].
1197
All of the above mentioned qualities justify using the NCA. This
algorithm straight maximizes a stochastic variant of the leave-one-
out k-nearest neighbors (KNN) score on the training set. Moreover,
It can train a low-dimensional linear projection of data used for
data visualization and fast classification [26]. In NCA, the impor-
tance of each feature is calculated based on its role in the prediction
of output [25]. However, since the real data distribution is un-
known, the algorithm attempts to optimize the performance based
on training data. The algorithm is restricted to learning quadratic
distance metrics. It can always be represented by symmetric posi-
tive semi-definite matrices. It is denoted by a transformation ma-
trix A, a metric is effectively learned as equation (2).
dðx; yÞ ¼ ðx  yÞ > Q ðx  yÞ ¼ ðAx  AyÞ > ðAx  AyÞ (2)
The goal is to reduce f(A), which is defined by equation (3).
XX X
f ðAÞ ¼ pij ¼ pi (3)
i j2Ci i
Since the NCA works based on random locations in the numeral
space, its results may be varied in the features ranking. Therefore, in
order to eliminate bias implied by random location start, the NCA
process has been repeatedly performed with a high number of it-
erations (n ¼ 1000) and the mean of values reported as the result of
NCA.
Validation
The two feature selection methods have been individually
validated. Data samples in the first method (combination of PSO
and ANN) at each run of the neural network are grouped and
stacked in a cross-validation scenario with k ¼ 5 in the training and
testing of the model. In order to eliminate bias caused by the
random start in the NCA problem space, feature selection was
performed using a large number of rounds (iteration ¼ 1000). The
mean of the importance values was considered as the result of this
method in term of importance of the features.
Functional analysis of the miRNAs from final model
Kaplan Meier plotter
Kaplan Meier plotter is an online tool capable of assessing the
impact of 54k genes and miRNAs on survival in 21 cancer types. The
system incorporates gene chip and RNA-seq data - sources for the
databases including GEO, EGA, and TCGA. The primary purpose of
the tool is a meta-analysis based discovery and validation of critical
biomarkers [27]. For a better understanding of the associations of
the considered miRNAs to the prognosis of PDAC, the KM plotter
was used. In miRNA section, the pan-cancer option was selected,
and the search was restricted to Pancreatic ductal adenocarcinoma
(n ¼ 178). The following criteria were used: Survival: OS, Auto
select best cutoff: checked, Follow up threshold: 240 months,
censor at threshold: checked.
Target genes prediction
MiRDIP target gene prediction online tool v.4.1 was used in or-
der to find the highest ranked probable target genes for each
miRNA in the joint final model. By combining different target gene
prediction algorithms, miRDIP is able to predict approximately 152
million human microRNA-targets, which are gathered from 30
other softwares, and specifies an integrative score for any of those
predicted target genes [28]. The provided integrative confidence
score is considered for classifying the predicted target genes as
“very high”, “high” and “medium” sections. In this study, the con-
fidence score 0.3 was set as the cut-off line in order to select the
predicted target genes for more functional evaluations.
1198 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204

Fig. 2. General form of an artificial neural network.

External validation
An independent cohort was developed as a validation set, con-
sisting of serum miRNA expression profiles from patients with
pancreatic cancer and healthy controls. The subjects were chosen
from two other GEO datasets (GSE112264 & GSE124158) consisting
of PC serum miRNA profiles and healthy controls (70 controls and
81 PC). The data of validation set samples were pre-processed then
injected into the trained model. Based on the evaluation of the
network in the prediction of the mentioned data, the confusion
matrix was analyzed.

Results

Differentially expressed miRNAs (DEMs)

A total of 1346, 1471, 127 and 93 miRNAs showed significant up/

down regulation in GSE106817, GSE113486, GSE85589 and
GSE59856 microarray datasets, respectively. After integration of
the results, 27 DEMs showed combined log-FC ±2 with lowest p-
values (Table 1).

Table 1
Top differentially expressed miRNAs through all analyzed datasets.

miRNA Combined Log-FC P-val

hsa-miR-125a-3p 0.457772533 7.68E-81

hsa-miR-8073 0.398337882 9.79E-80
hsa-miR-1238-5p 0.500430762 2.12E-78
hsa-miR-6893-5p 0.222837086 3.94E-60
hsa-miR-1290 0.251870064 1.36E-59
hsa-miR-4530 0.252766227 3.12E-52
hsa-miR-663a 0.217248906 3.12E-52
hsa-miR-1469 0.352761675 2.76E-47
hsa-miR-92a-2-5p 0.242443823 5.66E-46
hsa-miR-125b-1-3p 0.25596432 4.45E-45
hsa-miR-1236-5p 0.262190493 9.19E-43
hsa-miR-5100 0.247321172 2.51E-42
Fig. 1. Selection general procedure. hsa-miR-6075 0.257962134 6.24E-41
hsa-miR-6789-5p 0.282952299 1.34E-38
hsa-miR-7852 0.218510073 1.41E-38
hsa-miR-4536 0.285274858 4.34E-38
MiRNA-mRNA & PPI network construction hsa-miR-4490 0.308425262 5.13E-38
hsa-miR-3125 0.502933182 7.25E-38
STRING database v.9.0.5 was used to analyze the interactions hsa-miR-4476 0.242321912 5.93E-37
between the target genes of selected miRNAs [29]. Confident hsa-miR-575 0.226364357 7.39E-37
interaction score was set on 0.7. The protein-protein interaction hsa-miR-4736 0.209781341 2.42E-36
(PPI) networks were uploaded and visualized by Cytoscape hsa-miR-532-5p 0.235895661 4.16E-36
hsa-miR-3910 0.238832069 4.30E-36
software. Another network consisting of the interactions be-
hsa-miR-3927 0.230507438 6.91E-36
tween the selected miRNAs and their predicted target genes were hsa-miR-134-3p 0.215457081 2.06E-34
constructed and merged to the PPI network by the use of Cyto- hsa-miR-3128 0.201005266 2.82E-34
scape [30]. hsa-miR-4696 0.246605902 5.07E-34
 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204 1199

Machine learning of PDAC based on selected features (miRNA signatures) were

analyzed. Fig. 3 c illustrates the result of the intersection of two
The machine learning process for PC consists of three stages in feature selection methods. The final model comprises of the most
which different data mining techniques are used. The results of promising potential miRNAs (miR-663a, miR-1469, miR-92a-2-5p,
these techniques are presented in this section. miR-125b-3p and miR-532e5p).

Feature selection PSO þ ANN

For selecting features to build the model, two methods were
employed. First, more important features in both methods were Fig. 3 a shows the selected features using PSO and ANN. The
selected and evaluated; then, the selected features were combined results show that combination of PSO and ANN selected 14 miRNAs
and analyzed. Finally, the performance of the ANN in the prediction (including miR-125a-3p, miR-663a, miR-1469, miR-92a-2-5p, miR-

Fig. 3. Combination of different feature selection methods (PSO þ ANN and NCA) a) PSO þ ANN Feature selection result, b) NCA values means over iterations, and c) final set of
feature selection, the intersection of two feature selection paths.
1200 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
125b-1-3p, miR-5100, miR-7852, miR-4536, miR-4490, miR-3125,
miR-532e5p, miR-3927, miR-3128 and miR-4696) as the most
powerful representative, to diagnosis of PDACs from healthy
controls.
NCA
Fig. 3 b illustrates results of NCA iterations in the form of mean
calculation for NCA feature importance values over 1000 iteration.
The results show that 8 miRNA signatures such as miR-8073, miR-
6893, miR-4530, miR-663a, miR-1469, miR-92a-2-5p, miR-125b-
1e3 and miR-532e5p were selected by NCA.
Cross-validation
In order to validate the results, and prevent any biases that
might be implied in, validation of data analysis results was per-
formed as below:
The first path of feature selection (PSO þ ANN) was done in an
iterative manner as the PSO routine which is an evolutionary
optimization technique. Furthermore, the performance of ANN in
classification of PDAC based on important miRNA signatures was
evaluated. Fig. 4 shows the error of ANN training, validation and
testing. As shown, after a few epochs (about 42) error gradient
Fig. 4. Error of ANN in PC classification task (train, validation and test).
Fig. 5. Survival curves for high ranked miRNAs.
(difference between two successive training epoch errors) shows a
flattened manner, moreover the error value converged. This in-
dicates that ANN didn’t experience over-fitting situation and the
findings are reliable. On the other hand, in order to avoid biases in
the second path (NCA) as stated in the method section, rounds of
NCA executions were done to achieve more accurate and reliable
results (number of iterations ¼ 1000).
MiRNAs associated to overall survival of PDAC
Survival analysis of the top-ranked miRNAs from the joint model
was done using Kaplan-Meier plotter. 3 of 5 miRNAs with high ranks
in final model, hsa-mir-1469, has-mir-663a and has-mir-532 were
significantly associated with the prognosis of patients with PDAC.
These three miRNAs obtained P-values of 0.001, 0.001 and 0.009,
respectively, and hazard ratios of 2.27, 2.27 and 0.59, respectively,
between the high and low expression groups. Fig. 5A, B and C
represent the Kaplan-Meier survival curves for those three miRNAs.
Target genes of the joint model miRNAs
Overall 1312 target genes for three up-regulated miRNAs (miR-
532, miR-1469 and miR-663a) and 309 for the two down-regulated
miRNAs (miR-125b-1-3p and miR-92a-2-5p) were predicted.
PPI & miRNA-mRNA networks
The results of integrating constructed PPI network of predicted
target genes and the miRNA-mRNA interaction networks in Cyto-
scape for down-regulated (Fig. 6) and up-regulated (Fig. 7) miRNAs
separately, showed that miR-92a-2-5p and miR-125b-1-3p as
down-regulated miRNAs have two possible target genes in com-
mon. PEA15 and FOXP4 are targeted by both miRNAs in this
network. The interaction network of up-regulated miRNAs (miR-
1469, miR-663a and miR-532) demonstrated that these three
miRNAs target a module consisting of 45 predicted target genes.
External validation
Based on the evaluation of the network in the prediction of the
validation data, the confusion matrix was reported as follows: Ac-
curacy: 0.93, Sensitivity: 0.93 and Specificity: 0.92. (Fig. 8).
 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
Fig. 6. The interaction network of down-regulated high ranked miRNAs and their predicted target genes. Two predicted target genes (PEA15 and FOXP4) are in common between
two miRNAs.
Discussion
PDAC is one of the most mortal cancers and a big health problem
worldwide. The main challenge for diagnosis and treatment of this
cancer is the late detection of the patients due to the late mani-
festation of symptoms and limited efficacy of current diagnostic
methods. Accordingly, a variety of diagnostic and prognostic bio-
markers have been proposed, but most of them have failed to be
applicable to clinical plans. In recent years, microRNA-based liquid
biopsy (miRNAs) has become a promising area of research for early
diagnosis of malignancies. Several studies have also found miRNAs
in plasma specimens of PC patients to be abnormally expressed,
indicating that circulating miRNAs could be useful for PC diagnosis
[31]. For example, for diagnosis of PC different stages, Ganepola
et al. utilized a panel comprised of three miRNAs (miR-22, miR-
642b-3p, and miR-885-5) in plasma and the AUC value was 0.97
for discrimination of the PC cases [32]. In another study, Liu R et al.
used a serum panel consisting of miR-20a, miR-21, miR-24, miR-25,
miR-99a, miR-185, and miR-191 for the detection of stage I-IV PCs,
and the AUC value was 0.99 [33]. What should be considered is that
top biomarkers, even those acquired by highly advanced methods,
have not reached a better diagnostic and prognostic performance
than current biomarkers in large-scale clinical trials [34]. Through
recent decades, advances in the field of computational biology in
cancer omics-based research (such as machine learning methods)
have enabled researchers to evaluate more powerful diagnostic
biomarkers and models for PC [35]. In this study by analyzing on-
line microarray miRNA expression profiles from GEO (Gene
Expression Omnibus) database, we have determined a set of
circulating miRNAs of patients with pancreatic cancer, as a poten-
tial diagnosis model.
1201
This section reviews the result of this study from a variety of
perspectives:
Techniques used in feature selection
Using a combination of Artificial Intelligence techniques, we
obtained a valid analysis on the ordering of features importance,
and as the results of the study show, the Artificial Neural Network
as a simulation of human brain learning scenario with Particle
Swarm Optimization as a simulation of the collective decision-
making process in the nature lead to an appropriate way of
feature selection. On the other hand, features ranking based on the
use of the NCA iterations, a pure math-based approach, reinforces
the findings of the first path and confirms them. Combining the
results of two paths leads to more reliable results mathematically,
as well as practically as follows. In fact, we used an unbiased data-
driven technique consisting of microarray miRNA transcriptomic
data integrated with machine learning approaches to investigate
and confirm potential biomarkers together as diagnostic models for
PDAC detection.
Advantages and disadvantages of the methods used
First, the types of feature selection methods (filter based,
embedded and wrapper) were described: The filter methods pick
up the intrinsic properties of the features (i.e., the “relevance” of the
features) measured via univariate statistics instead of cross-
validation performance. Wrapper methods (such as PSO þ ANN)
measure the “usefulness” of features based on the classifier per-
formance. Embedded methods, are quite similar to wrapper
methods since they are also used to optimize the objective function
1202 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
Fig. 7. The interaction network of up-regulated high ranked miRNAs. The genes with green color represent genes with tumor-suppressive functions.
or performance of a learning algorithm or model (such as ANN).
Wrapper methods are essentially solving the “real” problem
(optimizing the classifier performance), but they are also compu-
tationally more expensive in comparison to the filter methods due
to the repeated learning steps and cross-validation. Given the high
processing power of today’s hardware, the processing burden
imposed on this method is bearable and is not problematic in non-
super-sized data (such as ours).
Biological analysis of results
At the first step, a set of 27 miRNA signatures that were captured
through differential expression analysis as the most significant
markers for discriminating PDAC from healthy controls, were used
as the input data for two independent feature selection algorithms
(consisting of PSO þ ANN & NCA). We used two different methods
in order to increase the stringency of the work to find the most
promising model throughout all the input features. Combining the
results of those algorithms, we reached a final model consisting of 5
miRNAs (miR-663a, miR-1469, miR-92a-2-5p, miR-125b-3p and
miR-532e5p) with the greatest combinatorial AUC score in differ-
entiation PC from controls. For validation, we checked the impor-
tance and association of the selected miRNAs from the final model
to the overall survival rates of PC patients using another online
database named KM Plotter. The results showed that 3 miRNAs
amongst the final model (hsa-mir-1469, has-mir-663a and has-mir-
532) are comprehensively associated with the overall survival of
the PDAC patients based on their up or down-regulated expression
patterns. Next, for more evaluations of the functions and roles of
considered miRNAs, we performed target genes prediction and
functional enrichment analysis as well as PPI network construction
for those target genes. Finally, two miRNA-target genes interaction
networks were constructed for up and down regulated miRNAs
separately. The results are further discussed as follows:
Mir-1469, has shown aberrant expression in a variety of cancers
including pancreatic cancer [36e38]. Despite most of the studies
have reported down-regulation of miR-1469 in other cancers, in
this study we have found a significant up-regulation of this gene for
PDAC. The result of Overall Survival analysis also showed that the
patients with high expression of miR-1469 have a significantly
poorer prognosis in comparison to the healthy group (Fig. 5A).
Similarly, MiR-663a which was amongst the up-regulated miRNAs
in our final model has yielded a controversy about its expression
levels in different types of cancers including PC. Some studies re-
ported down-regulation of this miRNA in serum samples of Gastric,
Colorectal, Hepatocellular and non-small cell lung cancers [39,40];
but some others reported up-regulation of these gene and
appointed an oncogenic function for this gene in a group of cancers
[41,42]. The results of our analyses of data from 671 PC serum
samples and healthy controls showed that the level of this miRNA is
 B. Alizadeh Savareh et al. / Pancreatology 20 (2020) 1195e1204
Fig. 8. The performance of the final model in diagnosis of PC patients from healthy controls in the validation set.
significantly up-regulated in PC in comparison to the control group.
This pattern is meaningfully in line with the Overall Survival
analysis results that we performed for this gene, where it was
demonstrated that high levels of miR-663a are associated with
poorer prognosis of PDAC patients. The same situation is true for
miR-532 and this gene shows diversity in expression in different
types of cancers [43e45]. Regarding PC, there is no report of de-
regulation of this gene in literature but the results of our evalua-
tions on expression data of serum of PC patients represent a distinct
up-regulation of this gene. However, the KM plotting showed that
PDAC patients with decreased expression of this gene have a
shorter life span and poorer survival. For all these 3 up-regulated
miRNAs of our final model, we performed target genes prediction
and miRNA-miRNA interaction analysis. The results showed that
these 3 miRNAs may regulate a module consisting of 45 target
genes. Functional evaluations revealed that some critical tumor
suppressors such as CDKN2A and RHOB exist amongst the target
genes of those miRNAs.These findings suggest that the considered
miRNAs may get associated with PC tumorigenesis via inhibiting
those tumor suppressor genes, however, the existence of such as-
sociations needs further experimental validations. On the other
hand, an interaction network was also constructed for down-
regulated miRNAs and their target genes. MiR-92a-2-5p and miR-
125b-1-3p demonstrated to have 2 target genes in common,
PEA15 and FOXP4. For PEA15 gene, which is a well-known ERK
signaling regulator with anti-proliferation effects [39], there does
not seem to have any associations with the down-regulation of
miR-92a-2. But the other gene, FOXP4 has been reported as a
modulator of tumorigenesis by several studies [40,41]. However, to
infer the correlations among any of the discussed miRNAs and the
target genes with PC, experimental evaluations are needed. Hence,
although some additional biomarkers as a diagnostic model were
identified in this bioinformatics and machine learning study for
potential consideration in future research, the outputs of these
analyses do not support the immediate clinical use of these bio-
markers without further comprehensive testing in broad case-
control and cohort studies.
Conclusion
This study aimed to find a robust diagnostic model for pancre-
atic cancer through machine learning methods, using miRNA
1203
biomarkers that were handed by a comprehensive bioinformatics
data mining approach. Our results established an index including
clinically-chosen miRNAs as biomarkers for pancreatic cancer
diagnosis and demonstrated that by ranking miRNAs via feature
selection methods, the top discriminating miRNAs for pancreatic
cancer detection among those bioinformatically verified can be
acquired. Nonetheless, these findings have to be clinically
confirmed.
Author contribution
RS designed the study, did the bioinformatics analyses and
wrote the manuscript. BA performed the Machine learning analyses
and helped writing manuscript. AB and AB helped improving il-
lustrations and data collection. HA, AS and MZ were the clinical
counselors expert in pancreatic cancer and clarified the main goals
of the project and interpreted the data. SS edited the manuscript in
English writing and scientific aspects.
Declaration of competing interest
The authors declare they have no conflict of interests.
Acknowledgments
We thank our colleague, Dr. Golnaz Bahramali, Department of
Hepatitis and AIDS, Pasteur Institute of Iran, who provided insight
and expertise that greatly assisted the research, although she may
not agree with all of the interpretations of this paper.
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