Exosomal microRNAs as potential circulating biomarkers in gastrointestinal tract cancers: a systematic review protocol
Gheytanchi et al. Systematic Reviews
Exosomal microRNAs as potential circulating biomarkers in gastrointestinal tract cancers: a systematic review protocol
Elmira Gheytanchi 2
Zahra Madjd 1
Leila Janani 0
Arezoo Rasti 2
Roya Ghods 6
Fatemeh Atyabi 5
Mohammad Hossein Asadi Lari 4
Sadegh Babashah 3
0 Department of Biostatistics, School of Public Health, Iran University of Medical Sciences , Tehran , Iran
1 Oncopathology Research Center, Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences , Tehran , Iran
2 Oncopathology Research Center, Iran University of Medical Sciences , Tehran , Iran
3 Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University , Tehran , Iran
4 Department of Cellular and Physiological Sciences, Faculty of Medicine, University of British Columbia (UBC) , Vancouver, BC , Canada
5 Department of Pharmaceutics, Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences , Tehran , Iran
6 Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Oncopathology Research Center, Iran University of Medical Sciences , Tehran , Iran
Background: Metastasis is the most frequent type of recurrence in gastrointestinal (GI) cancers, and there is an emerging potential for new diagnostic and therapeutic approaches, especially in the cases of metastatic GI carcinomas. The expression profiles of circulating exosomal microRNAs are of particular interest as novel non-invasive diagnostic and prognostic biomarkers for improved detection of GI cancers in body fluids, especially in the serum of patients with recurrent cancers. The aim of this study is to systematically review primary studies and identify the miRNA profiles of serum exosomes of GI cancers. Methods and design: This systematic review will be reported in line with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidance. Relevant studies will be identified through a comprehensive search of the following main electronic databases: PubMed, Web of Science, Embase, Scopus, and Google Scholar, with no language restrictions (up to July 2017). Full copies of articles will be identified by a defined search strategy and will be considered for inclusion against pre-defined criteria. The quality assessment of the included studies will be performed by the Newcastle-Ottawa Scale (NOS). Data will be analyzed using Stata software V.12. Publication bias will be assessed by funnel plots, Beggs' and Eggers' tests. The levels of evidence for primary outcomes will be evaluated using the GRADE criteria. Discussion: The analysis of circulating exosomal miRNA profiles provides attractive screening and non-invasive diagnostic tools for the majority of solid tumors including GI cancers. There is limited information regarding the relationship between serum exosomal miRNA profiles and the pathological condition of patients with different GI cancers. Since there is no specific biomarker for GI cancers, we aim to suggest a number of circulating exosomal miRNA candidates as potential multifaceted GI cancer biomarkers for clinical utility. Systematic review registration: PROSPERO CRD42017057129
Circulating; Exosomal microRNAs; Gastrointestinal tract; Cancer
Gastrointestinal tract (GI) cancers represent a major
challenge to healthcare systems given that they
constitute 25% of cancer-related mortalities [
]. The incidence
rate of GI cancers varies between developed and
developing countries [
]. Among the 15 most frequent
malignancies worldwide, one out of four new cancer
cases and one out of three annual cancer-related deaths
are attributed to GI cancers [
miRNAs are small non-coding RNA molecules
consisting of approximately 22 nucleotides that control gene
expression through targeting specific mRNAs bearing
partially complementary target sequences for
degradation and/or translational repression. miRNAs have been
shown to be involved in regulating several cellular
processes including cell differentiation, proliferation, and
apoptosis in both normal and pathological conditions [
]. miRNAs can act as either oncogenes or tumor
suppressor genes in the networks specifically altered during
cancer development and progression [
4, 6, 7
]. They are
present as mobile genetic components for cell to cell
communication or through the systemic circulation
system within the body [
Exosomes are nano-sized membrane vesicles, ranging
from 30 to 100 nm in size, that are secreted by several cell
types, including cancer cells, by exocytosis and can be
isolated from different body fluids and malignant ascites [
]. Exosome content is composed of unique miRNAs,
mRNA, DNA, lipids, and proteins that indicate the genetic
information of their parental cells [
]. Exosomes play an
important role in transferring bioactive oncogenic cargo
to non-transformed recipient cells which cause tumor
invasion, drug resistance, metastasis, and modulation of
cancer metabolism [
]. Findings have shown that the
discovery of specific exosomal content and markers may
represent a novel diagnostic tool [
]. Since the
majority of serum miRNAs are encapsulated in exosomes in
order to be more stable, the exosomal miRNAs could be
applied as promising non-invasive biomarkers and
potential targetable factors in cancer diagnosis and treatment
9, 14, 15
]. Recent studies have focused on
“tumor-specific” or “tumor-enriched” miRNAs which are specifically
packaged into exosomes [
Exosomal miRNA profiling of serum from different
cancer patients versus healthy controls has revealed
significant differences in relation to cancer development and
metastasis, indicating a possible use of these miRNAs as
prognostic biomarkers [
].The role of specific exosomal
miRNAs as biomarkers in cancer diagnosis, prognosis,
and screening have been studied in several types of
cancers, including GI cancers [
Some of the serum exosomal miRNAs can be used as
biomarkers to predict the recurrence of GI carcinomas
such as hepatocellular cancer and esophageal squamous
cell carcinoma [
]. The diagnostic and prognostic
roles of exosomal miRNA profiles of colorectal cancer
(CRC), as the most common malignancy of the GI system,
have been evaluated in several studies [
15, 31, 35–40
Previous studies had been developed to investigate the
preclinical and clinical characteristics of GI cancers and
have led to improved overall and progression-free
survival rates. However, there is an emerging potential for
screening, diagnosis, or surveillance of cancer and the
designing of new therapeutic approaches, especially in
the cases of metastatic GI carcinomas. The identification
and validation of biomarkers that can be measured
routinely in easily accessible samples, including plasma, and
that are able to diagnose cancer and predict treatment
efficacy and the risk of progression or relapse is a major
challenge in cancer research. To date, many researchers
have published their data on the clinical relevance of
micoRNAs and exosomal miRNA expression and
circulating exosomal miRNAs in different clinical and
nonclinical samples including tissue, stool, serum, and cell
lines of GI cancers [
15, 31, 35, 40–44
Recently, a published review showed that the exosomal
miRNAs could be detected and isolated from body fluids
such as saliva. This review primarily provided an overview
on exosomal miRNAs as diagnostic markers of GI cancers
with a focus on the origin and trafficking of exosomes
between cells and techniques to isolate exosomal miRNAs,
micoRNAs, and exosomal miRNAs expression [
However, there is currently no systematic review on circulating
exosomal miRNAs as promising non-invasive biomarkers
for GI cancers. In the current systematic review, we focus
on studies that have evaluated circulating exosomal
miRNAs as non-invasive biomarkers in serum of patients with
primary GI tumors. We also aim to carry out a systematic
review of published exosomal miRNAs, profiling studies
that were performed by microarray and real-time PCR
methods and then compare the exosomal miRNA
expression profiles between primary and metastatic tumors in
the serum or plasma of GI cancer patients. Additionally,
we intend to identify the most consistently downregulated
and upregulated circulating exosomal microRNAs in GI
cancers and introduce some of them as diagnostic and
prognostic biomarkers for non-invasive clinical
applications. Although identification of exosomal miRNAs in
other clinical specimen may be significant, in practice, it is
difficult to apply invasive methods on patients. On the
other hand, it would be challenging to apply findings
about exosomal miRNAs expression in non-clinical
samples into clinical contexts. Therefore, this systematic
review aims to evaluate the circulating exosomal miRNAs as
promising non-invasive biomarkers of GI cancers, for the
Aim of the study
The primary objective of this review is to provide a
systematic review of primary studies to estimate the
frequency of exosome-encapsulated miRNAs in the
circulating blood of patients with GI cancers. The
secondary objectives are to identify the expression pattern
(up- and downregulation) of exosomal miRNAs in
patients with different gastrointestinal tract cancers and to
identify the specific circulating exosomal miRNAs
involved in the diagnosis and prognosis of GI cancers.
This review will complement the findings of an existing
review published in 2016 [
This review of studies should address the following points:
1. To establish the frequency of circulating exosomal
miRNAs in GI cancers.
2. To establish the up- and downregulated circulating
exosomal miRNAs in GI cancers.
3. To identify the specific circulating exosomal miRNAs
involved in the diagnosis and prognosis of GI cancers.
This review protocol has been published in the
PROSPERO International prospective register of systematic
reviews (http://www.crd.york.ac.uk/PROSPERO), with the
registration number CRD 42017057129. In addition, the
completed review will be reported in line with the
Preferred Reporting Items for Systematic Reviews and
MetaAnalyses (PRISMA) guidance [
] (Additional file 1).
Criteria for considering studies for the review
Observational studies (cross-sectional, case-control, and
cohort studies) investigating circulating exosomal
miRNAs in diagnosis and prognosis of GI malignancies,
including upper and lower GI, and hepatopancreato-biliary
will be included.
The participants are patients with GI cancers.
Types of studies to be excluded
Studies assessing in vitro and in vivo models, reviews,
letters, editorials, case reports, and case series studies
will be excluded.
Search methods for identification of studies
The search strategy for identification of relevant studies
will comprise the following main electronic databases:
PubMed, Web of Science, Embase, and Scopus, as well
as Google Scholar search engines with no language
restrictions (up to July 2017). An example of the PubMed
search strategy is shown in Table 1. The search syntax
will be modified in other databases accordingly.
Reference lists of relevant primary studies, reviews, and
key journals will be searched for additional studies.
Screening of the studies
Duplicate studies will be removed. All databases will be
searched, and the titles and abstracts will be extracted
(EGh). All the retrieved titles and abstracts from the
electronic search will be screened, and the relevant data
will be independently extracted (EGh and AR) according
to the previously described inclusion criteria. The
extracted data will then be cross-checked to rule out any
discrepancies. Unresolved discrepancies will be referred
to a third author to solve (LJ). All the reasons for
exclusion of ineligible studies will be recorded.
Data extraction (selection and coding)
Data for each of the included studies will be extracted as
1. Study characteristics (first author’s surname,
publication year, publication language, country,
study design, setting, locations, criteria for
sample selection and sample size, diagnostic
criteria, outcomes measured, patient enrolment
strategies, exosome isolation and identification
methods, miRNA profiling, real-time PCR results
of serum samples)
2. Participants’ characteristics (age, gender)
3. Frequency estimates of expression of circulating
Discrepancies between two reviewers will be resolved
by consensus. An independent investigator will be
consulted through discussion to reach consensus where
there is uncertainty or disagreement between reviewers.
#1 AND #2 AND #3
The asterisk (*) is used to signify truncation
(mesentery[Mesh] OR stomach[Mesh] OR Pancreas[Mesh] OR rectum[Mesh] OR duodenum[Mesh] OR jejunum[Mesh] OR ileum[Mesh] OR
cecum[Mesh] OR colon[Mesh] OR mouth[Mesh] OR esophagus[Mesh] OR Gastrointestinal Tracts[Mesh] OR “Gastrointestinal Tracts”[tiab] OR
“GI Tract”[tiab] OR “GI Tracts”[tiab] OR “gastrointestinal tract”[tiab] OR “Gastrointestinal Cancer”[tiab] OR “Digestive Tract”[tiab] OR “Digestive
Tracts”[tiab] OR “Lower GI Tract”[tiab] OR GI[tiab] OR oral[tiab] OR mouth[tiab] OR esophagus[tiab] OR gullet[tiab] OR gastric[tiab] OR
duodenum[tiab] OR jejunum[tiab] OR ileum[tiab] OR cecum[tiab] OR colon[tiab] OR colorectal[tiab] OR sigmoid[tiab] OR rectum[tiab] OR
anus[tiab] OR mesentery[tiab] OR hepatic[tiab] OR liver[tiab] OR hepatocellular[tiab] OR stomach[tiab] OR Pancrea*[tiab])
(Neoplasms [Mesh] OR Cancer*[tiab] OR Neoplasm*[tiab] OR Carcinoma[tiab] OR Tumo*[tiab])
(extracellular vesicles[Mesh] OR exosomes[Mesh] OR exosome*[tiab] OR “extracellular vesicle”[tiab] OR “extracellular vesicles”[tiab] OR
microvesicle[tiab] OR “Shedding Microvesicles”[tiab])
Corresponding authors will be contacted if further
information is needed. If no response is received after
sending a reminder, studies will be excluded.
Risk of bias (quality) assessment
Quality assessment of observational studies is challenging
due to their methodological complexities and the
subjective nature of quality evaluations [
]. The Cochrane
Collaboration endorsed the use of the Newcastle-Ottawa
Scale (NOS) to assess the quality of observational studies
in its 2011 handbook [
]. The final risk of bias of
included studies will be performed by the above mentioned
tool. Two reviewers will independently assess the
methodological quality of primary studies by the NOS. This
tool is used for assessing the quality of non-randomized
studies included in a systematic review and/or
metaanalyses. The method was developed as a collaboration
between the University of Newcastle, Australia, and the
University of Ottawa, Canada, using a Delphi process to
define variables for data extraction. Using the tool, each
study is judged on eight items, categorized into three
groups: the selection of the study groups, the
comparability of the groups, and the ascertainment of either the
exposure or outcome of interest for case-control or cohort
studies respectively . Separate tools were developed
for cohort and case-control studies. It has also been
adapted for cross-sectional studies [
]. For more
details, see three appendix scales for evaluating the quality
assessment of the above mentioned observational studies
(Additional files 2, 3, and 4).
Strategy for data synthesis
Given the considerable variability among cancers and
the difficulty in detecting common exosomal miRNAs
and characteristics of the studies, the extracted
outcomes will be summarized and reported using
descriptive statistics without conducting any meta-analyses.
Two separate tables will be used in all included studies.
The first table will provide details on study quality
according to the mentioned tool, and the other one will
include study design, participants, and frequency of
exosomal miRNAs in patients with GI cancers. The
publication bias will be assessed by funnel plots (i.e., plots of
study results against precision) and Beggs’ and Eggers’
tests. We will assess the overall quality of the evidence
for each main outcome based on the GRADE (Grading
of Recommendations Assessment, Development and
Evaluation) system [
]. GRADE provides explicit
criteria that address study design, risk of bias,
imprecision, inconsistency, indirectness, and magnitude of the
effect to rate the quality of evidence across studies. This
method rates the quality of the evidence from high (very
confident that the true effect lies close to that of the
estimated effect) to very low (very little confidence in
the effect estimate).
Given the diversity of cancer types and different
reported exosomal miRNAs across the studies, we
anticipate that we cannot perform a meta-analysis; therefore,
no sensitivity analysis will be applicable to this study.
Analysis of subgroups or subsets
If the data is determined to be heterogeneous, we will
conduct a narrative synthesis of the findings from the
included studies; therefore, a subgroup analysis will not be
applicable to this study.
Emerging evidence supports the hypothesis that exosomal
microRNAs can be used as circulating diagnostic
biomarkers of several cancers. However, there is currently no
collective view of which circulating exosomal microRNAs
are suitable candidates. The distinctive properties of
miRNAs within exosomes can be manipulated to render them
suitable for the development of effective diagnostic and
targetable tools for non-invasive screening and targeting
of tumor cells in cancer patients. Reviews showing the
serum exosomal microRNAs in different cancers have
already emerged [
1, 39, 44
], but to date, this will be the
first systematic review to evaluate the circulating
exosomal miRNAs in the serum of patients with GI cancers.
The findings of this review may be used in clinical settings
and in designing and developing of the novel non-invasive
biomarkers for early diagnosis and pathological condition
of patients with GI cancers.
Additional file 1: PRISMA-P (Preferred Reporting Items for Systematic
Review and Meta-Analysis Protocols) 2015 checklist [
]. (DOC 85 kb)
Additional file 2: Newcastle-Ottawa quality assessment scale for
crosssectional studies. (PDF 189 kb)
Additional file 3: Newcastle-Ottawa quality assessment scale for
casecontrol studies. (PDF 17 kb)
Additional file 4: Newcastle-Ottawa quality assessment scale for cohort
studies. (PDF 93 kb)
GI: Gastrointestinal; microRNAs: miRNAs, miRs; PRISMA: Preferred Reporting
Items for Systematic Reviews and Meta-Analyses
Availability of data and materials
All recorded data from the data extraction process will be available on request
to the extent that they are not included in the systematic review article.
ZM, LJ, EGh, AR, and RGh contributed to the concept and study design. EGh
and AR developed the search strategies. EGh and LJ contributed to the
implementation and analysis of the studies and will also screen potential
studies and extract data and assess quality, while ZM, RGH, AR, FA, SB, and
MhAL will review the work. EGh and AR contributed to the initial drafting
and critical revision the manuscript, and all authors gave feedback to the
final draft of the protocol. EGh approved the manuscript for submission. All
authors read and approved the final manuscript.
Ethics approval and consent to participate
As this systematic review will only be based on published data already in the
public domain, an ethical approval is not required. The findings of the current
study will be disseminated through a publication in a peer-reviewed journal
and conference presentations. To the best of our knowledge, there are no
systematic reviews that have specifically looked at the frequency of
exosomeencapsulated miRNAs in the circulating blood of patients with GI cancers. We
will submit the findings of this systematic review for publication and presentation
at national and international conferences to update information on the circulating
exosomal miRNAs as candidates of GI cancers. This review will ultimately help
inform and guide future studies in the field.
Consent for publication
The authors declare that they have no competing interests.
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