BMJ Open · Hemmat Street (Highway), Next to Milad Tower, Tehran, Iran Post Code: 14496-14530 Email address: [email protected] Tel: +982186703212 Fax: +982188622608 Abstract Introduction:

For peer review only

Diagnostic and prognostic accuracy of miR-21 in renal cell carcinoma: A systematic review protocol

Journal: BMJ Open

Manuscript ID: bmjopen-2015-009667

Article Type: Protocol

Date Submitted by the Author: 07-Aug-2015

Complete List of Authors: Rasti, Arezoo; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of), Mehrazma, Mitra; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Madjd, Zahra; Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran, Keshtkar, Abbas; School of Public Health,Tehran University of, Department of Epidemiology and Health Statistics

Babashah, Sadegh; School of Biological Science,Tarbiat Modares University, Tehran, Iran, Department of Genetics Roudi, Raheleh; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of) Tehran,

<b>Primary Subject Heading</b>:

Oncology

Secondary Subject Heading: Pathology, Urology

Keywords: ONCOLOGY, Kidney tumours < ONCOLOGY, MOLECULAR BIOLOGY

For peer review only - http://bmjopen.bmj.com/site/about/guidelines.xhtml

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Diagnostic and prognostic accuracy of miR-21 in renal cell carcinoma: A systematic review

protocol

Arezoo Rasti1, Mitra Mehrazma*

2, Zahra Madjd1

1, 3, Abbas Ali Keshtkar

4, Sadegh Baba Shah

5

Raheleh Roudi1

Address for correspondence:

*Mitra Mehrazma, MD, Pathologist

Oncopathology Research Center

Iran University of Medical Sciences (IUMS)

Hemmat Street (Highway), Next to Milad Tower, Tehran, Iran

Post Code: 14496-14530

Email address: [email protected]

Tel: +982186703212

Fax: +982188622608

Abstract

Introduction: Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the important

unmet medical needs in RCC is prognostic biomarker enabling identification of patients at high risk

of relapse after nephrectomy. New biomarkers can help improve the diagnosis and hence the

management of renal cancer patients. Thus, this review aim to clarify the prognostic and diagnostic

accuracy of miR-21 in renal cell carcinoma patients.

Methods and analysis:

We will include observational studies evaluating the diagnostic and prognostic role of miR-21 in

patients with renal cancer the index test and reference standards should ideally be performed on all

patients. We will search PubMed, SCOPUS, and ISI web of sciences with no restriction of

language. The outcome will be the survival measures in adult patients with RCC.

Study selection and data extraction will be performed by two independent reviewers. Quality

assessment will be implemented using QUADAS-1. The publication bias and data synthesis will be

assessed by funnel plots and Begg’s and Egger’s tests using Stata software version 11.1.

Ethics and dissemination: No ethical issues are predicted. These findings will be published in a

peer-reviewed journal and presented at national and international conferences.

Registration number: This systematic review protocol is registered in the PROSPERO

International Prospective Register of Systematic Reviews, registration number (CRD42015025001).

Strengths and limitations of this study:

This systematic review, for the first time, will conduct to evaluate the prognostic and diagnostic

accuracy of Mir-21 in patients with RCC using comprehensive search of several databases without

restriction to languages.

The study screening, data extraction, and risk of bias assessment of the current study will be

conducted by two researchers independently.

We expect some potential heterogeneities between previous studies, including stage, and

histological grade in patient samples.

Introduction

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Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. (1)The incidence of

RCC is increasing annually about 20%.(2). Distinguishing RCC subtypes is of clinical importance

because they have different prognoses and subsequently different management plans(3).The most

common form of RCC is clear subtype (ccRCC) which accounts for 75% to 80% of cases. Other

less common subtypes include papillary(pRCC), chromophobe(chRCC)and collecting duct

carcinoma.(4) Although surgery is curative for localized disease, a significant proportion of patients

relapse or metastastasize. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic

RCC is dismal at ≤10%.(5).

The present absence of biomarkers for early detection and follow-up of the disease is responsible

for late diagnosis and subsequent poor prognosis(6)

Factors influencing RCC prognosis can be classified into anatomic, histologic, clinical and

molecular(2).Pathologic stage , based on the size and extent of invasion by the tumor ,is the most

accurate indicator of prognosis.(7).Recent evidence showed that molecular signature can classify

RCC subtypes more accurately than morphological characteristics (8-10). MicroRNAs are small

noncoding RNA nucleotides that post–transcriptionally regulate expression of their target proteins

(4)The first report suggesting a role of miRNAs in caner was published in 2002(11)

Evidence has shown that miRNAs are actively involved in kidney cancer pathogenesis (12, 13).

Potential roles of microRNAs as prognostic and diagnostic tools in RCC and its subtypes have been

reported in many studies.(4, 14-33).Among the large number of miRs, miR-21 has received the

specific attention because of its relationship with multiple cancer entities, it is up regulated in many

tumors including breast, lung , Colon, pancreas, prostate and stomach cancers(34-36) (37).The

effect of miR-21 on carcinogenesis and tumor progression has been experimentally

analyzed(38).MiR-21 regulates epithelial-mesenchymal transition phenotype and hypoxia-inducible

factor-1a expression in breast cancer stem cell-like cells and contribute to cell migration for

metastasis(39).The global predicting role of miR-21 for survival in patients with a variety of

carcinomas is summarized in a meta-analysis (40).

To date, many researchers have published their data for the prognostic value of miR-21 in Renal

cancer, and have raised concerns about the efficiency of miR-21 as a biomarker, whereas these

issue remain questionable. This systematic review, for the first time, will conduct to evaluate the

diagnostic and prognostic accuracy of miR-21 in RCC patients.

Objectives

Our primary objectives are to determine the diagnostic and prognostic accuracy of miR-21 in RCC

patients and the secondary objectives are determination the diagnostic and prognostic accuracy of

miR-21 in RCC subtypes and evaluation of the heterogeneity and its potential sources in primary

studies.

Methods Methods for this systematic review have been developed according to recommendations from the

PRISMA checklists and the PRISMA Flow Diagram will be used to describe the flow of

information through the different phases of the systematic review.(41)

The protocol of this systematic review has been registered on PROSPERO 2015 (registration

number: CRD42015025001).

Preferred Reporting Items for Systematic reviews and Meta-Analyses for Protocols 2015

(PRISMA-P 2015) have been used for the preparation and reporting of this protocol for the

systematic review.(42)

Criteria for considering studies for this systematic review

Types of studies

Criteria for considering studies for this review:

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We will include all observational studies (cross sectional, case control and cohort). We will exclude

studies assessing animal models, review articles, proceeding conferences and case series studies on

dead patients from RCC.

Participants

Adult patients in both sexes with diagnosis of RCC will be included. Tumor classification and stage

were diagnosed according to TNM system and 2004 WHO classification (43, 44).

Index test

MiR-21

MiRs are highly conserved small noncoding RNA strands, approximately 22nucleotides in length

that post transcriptionally regulate gene expression and appear to be modulators of urologic cancers

(45, 46).MiR-21 is one of the most frequently studied cancer-related miRNAs and dysregulated in

most cancers by acting as oncogene.(47)

In RCC several studies describe up regulation of miR-21 compared with healthy kidney(4, 38).

There is a significant difference in expression levels of miR-21 between RCC subtypes with highest

level in ccRCC and pRCC subtypes(4).MiR-21 levels are associated with higher stage and grade, so

its expression level can be used in as a diagnostic marker to distinguish RCC subtypes(4).The

expression level of miR-21 is correlated with 5-year survival and pathological stage in RCC(48).

Patients who are miR-21 positive have statistically shorter disease free and overall survival rates

(4)In the current study, Mir-21 detection in RCC tissues or serum will define based on Quantitative

Real-Time RT-PCR.

There is several steps for mir-21 detection in RCC tissues based on Quantitative Real-Time RT-

PCR.

1. Total RNA have been extracted from RCC samples (formalin-fixed, paraffin-embedded tissues or

serum) using Commercial kits according to the manufacturer’s protocol.

2. RNA concentrations and A260/280 ratio with a Nano Drop have been determined.

3. The resulting miRNA for quantitative Real Time PCR (qRT-PCR) have been retained and

miRNA expression with TaqMan MicroRNA Assays have been measured.

4. Reverse transcription of miR-21 with a specified amount of total RNA using the TaqMan

MicroRNA specific Reverse Transcription Kit have been Performed.

5. Cycling conditions for miR-21 according to manufacturer’s protocols have been chosen .

6. All reactions have been performed in triplicates.

7. Values of miR-21 to a small nuclear RNA as reference gene have been normalized.

8.Relative over- or under expression of miR-21 in patients samples have been compared to the

normal ones by the ∆∆Ct method assuming equal RNA concentrations and efficiency of qRT-PCR

have been completed.

Gold standards

Studies will be eligible for this review if clinical diagnostic criteria recommended by WHO

classification and TNM system of the renal tumors of the adults were used as primary reference

standards. The pathological diagnosis of RCC is recommended for all renal masses existent in

radical nephrectomy or biopsies .Because clinical practice guidelines are routinely updated, we will

use the criteria of reference standard that the primary studies adopted.

Survival measures will be used as reference standards for prognostic accuracy.

Target conditions

The target conition for diagnostic and prognostic value will be proven RCC and survival measures

respectively.

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Outcomes

The our primary outcomes will be all survival measures of RCC patients and the secondary

outcomes will be to determine prognostic accuracy of Mir-21 in different subtypes of renal

cancer, as well as, determination of the heterogeneity and its potential sources.

Search methods for identification of studies

Electronic searches

We will search the following databases: PubMed, Web of Sciences, Scopus, up to 31 July

2015).The searches will be refined using the bloom term “AND” between the topics of RCC and

index test (miR-21).

PubMed search strategy

MeSH tags were found in PubMed. The details of the PubMed database search strategy and syntax

is sequentially provided below.

1. miR21

2. miR-21

3. microRNA-21

4. miRNA-21

5. hsa-mir-21

7.1 or 2 or 3 or 4 or 5

8. Renal

9. Kidney

10.8 or 9

11.7 AND 10

(miR21 [tiab] OR microRNA-21[tiab] OR miRNA-21[tiab] OR hsa-mir-21[tiab] OR miR-21[tiab])

AND (kidney [tiab] OR renal [tiab])

Other resources

Reference lists of relevant primary studies, reviews and key journals will be searched for additional

studies.

Data collection and analysis

A data extraction form will be developed and study data will be assessed and extracted

independently by two reviewers, AR and RR.

Study selection is summarized in a PRISMA flow diagram (figure 1).

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Figure 1: Flow diagram of the study selection process based on PRISMA guidelines

The following data will be extracted from each included study:

1. Study characteristics (author, year of publication, study design, setting, locations and patient

enrollment strategies).

2. Test characteristics (test type, test condition, prespecified cutoff values, sampling protocol, and

criteria for test positivity)

3. Reference standards (histopathological diagnosis or criteria of the guideline used)

4. Participant characteristics (age, gender, ethnicity, disease type, tumor sizes and tumor stages)

5. Statistics for meta-analysis are: (true positives (TP), False positives (FP), true negatives (TN)

and false negatives (FN), Sensitivity, Specificity, Positive and Negative Likelihood Ratios and Area

under the Curve(AUC) for diagnostic value and Overall survival(OS) ,Disease Free survival

(DFS)Recurrence Free Survival(RFS) for prognostic values.

Information not available in the studies will be sought from authors by email. Any discrepancies

will be resolved by consensus between the two authors.

Records identified through

database searching (n= )

Additional records

identified through other

Records after duplicates removed (n= )

Records screened (n=) Records excluded (n= )

Full-text articles

assessed for eligibility

Full-text articles

excluded, with reasons

Studies included in

qualitative synthesis (n= )

Ide

nti

fica

tio

Scr

ee

nin

g

Eli

gib

ilit

y

Incl

uded

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Assessment of methodological quality

Methodological quality of primary studies will be assessed according to the revised tool for

QUADAS-1 quality assessment tool. (49)

The defined questions will be answered as yes, no, or unclear and the score of each article will be

calculated. Two authors (A.R and R.R) will initially complete the pilot QUADAS-1 items in three

studies. If poor agreement is noted we will refine the tool then the updated tool will be applied to

complete the assessment for all included studies. Disagreements will be resolved by consensus.

Assesment of heterogeneity

To investigate heterogeneity, we will include study design (prospective or retrospective and year of

publication), population characteristics (gender, ethnicity, age, disease types, and stage

distribution), test characteristics (cutoff value, test type, and number of tests per screening round),

and versions of reference standards as our study-level variables. We will test these study-level

covariates in the bivariate model in the common threshold or add them to the Rutter and Gatsonis

HSROC model to evaluate heterogeneity in test threshold, diagnostic accuracy, and the shape of

curves. The likelihood ratio test will be used to determine the statistical significance of the

covariates included in the models.

Assessment of reporting bias

The publication bias will be assessed by funnel plots (ie, plots of study results against precision)

and Begg’s and Egger’s tests.

Statistical analysis and data synthesis Descriptive analysis:

All included studies will be overviewed and will be presented in two separate tables. One table will

provide details on study quality according to the review-specific QUADAS-1 tool mentioned above.

The other table will include study design, participants, test characteristics, results, outcomes and

reference standards.The following test characteristics will be extracted into 2 × 2 tables(TP, FP, TN,

and FN)for all included studies, Study-specific estimations of sensitivity and specificity with 95%

confidence intervals will be displayed in forest plots using review Manager (Version 5.2). These

graphical displays will reveal the variations in accuracy among the studies and the different types

and brands of the index test.

Gathered data about outcomes such as Disease Free survival (DFS), Recurrence Free and Survival

(RFS), Hazard Ratio with 95% will be used for prognostic value of index test.

Inferential statistics:

All the included studies will be synthesis after systemic review. Study-specific Hazard Ratio,

Sensitivity and Specificity estimates will be pooled using a fixed-effects model if no significant

heterogeneity exists, otherwise a random-effects model will be applied. The extent of heterogeneity

across studies will be checked using the chi-squared test and I-squared test; p=0.10 and/or I2>50%

indicating significant heterogeneity. For evaluating source of heterogeneity, sub-group analysis and

meta-regression will be used.

The potential for publication bias was assessed using funnel plot, the Begg rank correlation

method, and the Egger weighted regression method. A p-value of ≤ 0.05 will be considered to

be statistically significant.

All of the mentioned analysis will be performed using STATA version 11.1 software. (StataCorp

LP, College Station, TX, USA).

Sensitivity analysis

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A sensitivity analysis will be conducted to test the impact of the results according to the

methodological quality items rated by the QUADAS-1 tool criteria of the reference standards.

We will implement sensitivity analyses to explore the impacts of methodological quality and

sample size on the robustness of review conclusions. Meta-analyses will be repeated after excluding

studies with lower methodological quality and studies with sample sizes much bigger than those of

other studies. Sensitivity analyses will be reported with a summary table and review conclusions

will be interpreted with concerns for comparisons between the two meta-analyses.

Subgroup analyses

Subgroup analysis will be conducted according to region, gender, quality, sample size and cancer

subtype.

Discussion

Our systematic review and meta-analysis will allow patients, clinicians, and researchers to

determine the diagnostic performance of miR-21 for detection of RCC, particularly early-stage

RCC, and the potential role of this marker in prognosis of RCC patients. This systematic review

will also help policy makers and guideline.

Contributors MM, ZM, RR and AK were involved in study design. AK and AR developed the search strategies.

AR and AK was involved in study design, implementation, and analysis. AR drafted the manuscript

of the protocol and RR revised it.

AR and RR will also screen the potential studies, extract data and assess quality. Any discrepancies


Competing interests

The authors declare no conflict of interest.

Funding

No.

Competing interests

None

Data sharing statement

All recorded data from the data extraction process will be available on request to the extent that it is

not included in the systematic review article.

Author affiliations

1. Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran

2. Department of Pathology, Faculty of medicine, Iran University of Medical Sciences, Tehran, Iran

3. Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of

Medical Sciences, Tehran, Iran

4. Department of Epidemiology and Health Statistics, School of Public Health,Tehran University of Medical

Sciences ,Tehran, Iran

5. Department of Genetics, School of Biological Science, Tarbiat Modares University, Tehran, Iran

Funding

No

Competing interests

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None




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39. Han M, Wang Y, Liu M, Bi X, Bao J, Zeng N, et al. MiR-21 regulates epithelial-mesenchymal

transition phenotype and hypoxia-inducible factor-1α expression in third-sphere forming breast cancer

stem cell-like cells. Cancer science. 2012;103(6):1058-64.

40. Fu X, Han Y, Wu Y, Zhu X, Lu X, Mao F, et al. Prognostic role of microRNA-21 in various

carcinomas: a systematic review and meta-analysis. European journal of clinical investigation.

2011;41(11):1245-53.

41. Liberati A, Altman DG, Tetzlaff J, Mulrow C, Gøtzsche PC, Ioannidis JP, et al. The PRISMA

statement for reporting systematic reviews and meta-analyses of studies that evaluate health care

interventions: explanation and elaboration. Annals of internal medicine. 2009;151(4):W-65-W-94.

42. Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, et al. Preferred reporting

items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev.

2015;4(1):1.

43. Lopez-Beltran A, Scarpelli M, Montironi R, Kirkali Z. 2004 WHO classification of the renal tumors

of the adults. European urology. 2006;49(5):798-805.

44. Srigley JR, Amin MB, Delahunt B, Campbell SC, Chang A, Grignon DJ, et al. Protocol for the

examination of specimens from patients with invasive carcinoma of renal tubular origin. Arch Pathol Lab

Med. 2010;134(4):e25-30.

45. Catto JW, Alcaraz A, Bjartell AS, White RDV, Evans CP, Fussel S, et al. MicroRNA in prostate,

bladder, and kidney cancer: a systematic review. European urology. 2011;59(5):671-81.

46. Bartel D, editor MicroRNAs: genomics, biogenesis, mechanism, and function. cell 2004, 116:

281-297. PubMed Abstract| Publisher Full Text OpenURL; 2014.

47. Kumarswamy R, Volkmann I, Thum T. Regulation and function of miRNA-21 in health and

disease. RNA biology. 2011;8(5):706-13.

48. Zaman MS, Shahryari V, Deng G, Thamminana S, Saini S, Majid S, et al. Up-regulation of

microRNA-21 correlates with lower kidney cancer survival. PLoS One. 2012;7(2):e31060.

49. Whiting P, Rutjes AW, Reitsma JB, Bossuyt PM, Kleijnen J. The development of QUADAS: a tool

for the quality assessment of studies of diagnostic accuracy included in systematic reviews. BMC medical

research methodology. 2003;3(1):25.

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PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) 2015 checklist: recommended items to

address in a systematic review protocol*

Section and topic Item No Checklist item

ADMINISTRATIVE INFORMATION

Title:

Identification 1a Identify the report as a protocol of a systematic review

Update 1b If the protocol is for an update of a previous systematic review, identify as such

Registration 2 If registered, provide the name of the registry (such as PROSPERO) and registration number

Authors:

Contact 3a Provide name, institutional affiliation, e-mail address of all protocol authors; provide physical mailing address of

corresponding author

Contributions 3b Describe contributions of protocol authors and identify the guarantor of the review

Amendments 4 If the protocol represents an amendment of a previously completed or published protocol, identify as such and list changes;

otherwise, state plan for documenting important protocol amendments

Support:

Sources 5a Indicate sources of financial or other support for the review

Sponsor 5b Provide name for the review funder and/or sponsor

Role of sponsor or funder 5c Describe roles of funder(s), sponsor(s), and/or institution(s), if any, in developing the protocol

INTRODUCTION

Rationale 6 Describe the rationale for the review in the context of what is already known

Objectives 7 Provide an explicit statement of the question(s) the review will address with reference to participants, interventions,

comparators, and outcomes (PICO)

METHODS

Eligibility criteria 8 Specify the study characteristics (such as PICO, study design, setting, time frame) and report characteristics (such as years

considered, language, publication status) to be used as criteria for eligibility for the review

Information sources 9 Describe all intended information sources (such as electronic databases, contact with study authors, trial registers or other

grey literature sources) with planned dates of coverage

Search strategy 10 Present draft of search strategy to be used for at least one electronic database, including planned limits, such that it could be

repeated

Study records:

Data management 11a Describe the mechanism(s) that will be used to manage records and data throughout the review

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Selection process 11b State the process that will be used for selecting studies (such as two independent reviewers) through each phase of the

review (that is, screening, eligibility and inclusion in meta-analysis)

Data collection process 11c Describe planned method of extracting data from reports (such as piloting forms, done independently, in duplicate), any

processes for obtaining and confirming data from investigators

Data items 12 List and define all variables for which data will be sought (such as PICO items, funding sources), any pre-planned data

assumptions and simplifications

Outcomes and prioritization 13 List and define all outcomes for which data will be sought, including prioritization of main and additional outcomes, with

rationale

Risk of bias in individual studies 14 Describe anticipated methods for assessing risk of bias of individual studies, including whether this will be done at the

outcome or study level, or both; state how this information will be used in data synthesis

Data synthesis 15a Describe criteria under which study data will be quantitatively synthesised

15b If data are appropriate for quantitative synthesis, describe planned summary measures, methods of handling data and

methods of combining data from studies, including any planned exploration of consistency (such as I2, Kendall’s τ)

15c Describe any proposed additional analyses (such as sensitivity or subgroup analyses, meta-regression)

15d If quantitative synthesis is not appropriate, describe the type of summary planned

Meta-bias(es) 16 Specify any planned assessment of meta-bias(es) (such as publication bias across studies, selective reporting within studies)

Confidence in cumulative evidence 17 Describe how the strength of the body of evidence will be assessed (such as GRADE)

* It is strongly recommended that this checklist be read in conjunction with the PRISMA-P Explanation and Elaboration (cite when available) for important

clarification on the items. Amendments to a review protocol should be tracked and dated. The copyright for PRISMA-P (including checklist) is held by the

PRISMA-P Group and is distributed under a Creative Commons Attribution Licence 4.0.

From: Shamseer L, Moher D, Clarke M, Ghersi D, Liberati A, Petticrew M, Shekelle P, Stewart L, PRISMA-P Group. Preferred reporting items for systematic review and

meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015 Jan 2;349(jan02 1):g7647.

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Figure 1: Flow diagram of the study selection process based on PRISMA guidelines

Records identified through

database searching (n= )

Additional records identified

through other sources (n= )

Records after duplicates removed (n= )

Records screened (n= ) Records excluded (n= )

Full-text articles assessed

for eligibility (n= )

Full-text articles excluded,

with reasons (n= )

Studies included in qualitative

synthesis (n= )

Ide

nti

fica

tio

n

Scr

ee

nin

g

Eli

gib

ilit

y

Inclu

de

d

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Journal: BMJ Open

Manuscript ID bmjopen-2015-009667.R1


Date Submitted by the Author: 22-Oct-2015

Complete List of Authors: Rasti, Arezoo; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of) Mehrazma, Mitra; Iran University of Medical Sciences (IUMS), Oncopathology Research Center; Iran University of Medical Sciences, Department of Pathology Madjd, Zahra; Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran; Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Department of Molecular Medicine

Keshtkar, Abbas; School of Public Health,Tehran University of Medical Sciences, Department of Health Sciences Education Development Roudi, Raheleh; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of) Tehran Babashah, Sadegh; Faculty of Biological Sciences, Tarbiat Modares University, Department of Molecular Genetics


Oncology

Secondary Subject Heading: Pathology, Urology, Diagnostics



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protocol


1,2, Zahra Madjd*

1,2,3, Abbas Ali Keshtkar

4, Raheleh Roudi

1,

Sadegh Babashah5


2. Department of Pathology, Iran University of Medical Sciences, Tehran, Iran 3. Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of


4. Department of Health Sciences Education Development, School of Public Health, Tehran

University of Medical Sciences

5. Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University,

Tehran, Iran

Correspondence to

[email protected] [email protected]

Abstract

Introduction:

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. One of the most

important unmet medical needs in RCC is prognostic biomarker enabling identification of patients

at high risk of relapse after nephrectomy. New biomarkers can help improve the diagnosis and

hence the management of renal cancer patients. Thus, this review aims to clarify the prognostic and

diagnostic accuracy of miR-21 in renal cell carcinoma patients.



patients with renal cancer, the index test and reference standards should ideally be performed on all

patients. We will search PubMed, SCOPUS, and ISI web of sciences with no restriction of

language. The outcome will illustrate the survival measures in adult patients with RCC.

Study selection and data extraction will be performed by two independent reviewers. Quality

assessment will be implemented using QUADAS-1. The publication bias and data synthesis will be

assessed by funnel plots and Begg and Egger’s tests using Stata software version 11.1.




International Prospective Register of Systematic Reviews, registration number =

CRD42015025001.


This systematic review, for the first time, will conduct to evaluate the prognostic and diagnostic

accuracy of mir-21 in patients with RCC through comprehensive search of several databases

without placing restrictions on languages.

The study screening, data extraction, and assessing risk of bias of the current study will be


We expect some potential heterogeneities between previous studies, including stage, and


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Introduction

Renal cell carcinoma (RCC) is the most common neoplasm of adult kidney. (1)The incidence of

RCC is increasing annually about 20 %.(2). Distinguishing RCC subtypes is of clinical importance

because they have different prognoses and subsequently different management plans (3).The most

common form of RCC is the clear subtype (ccRCC) which accounts for 75% to 80% of cases. Other

less common subtypes include papillary (pRCC), chromophobe(chRCC)and collecting duct

carcinoma.(4) Although surgery is curative for localized disease, a significant proportion of patients

relapse or metastastasize. Metastatic RCC is difficult to treat. The 5-year survival rate for metastatic

RCC is dismal at ≤10 %.(5).

The present absence of biomarkers for early detection and follow-up of the disease is responsible

for late diagnosis and subsequent poor prognosis (6)

Factors influencing RCC prognosis can be classified into anatomic, histologic, clinical and

molecular (2).Pathologic stage , based on the size and extent of invasion by the tumor ,is the most

accurate indicator of prognosis.(7).Recent evidence showed that molecular signature can classify

RCC subtypes more accurately than morphological characteristics (8-10). MicroRNAs are small

noncoding RNA nucleotides that post–transcriptionally regulate expression of their target proteins

(4)The first report suggesting a role of miRNAs in caner was published in 2002(11)

Based on the provided data miRNAs are actively involved in kidney cancer pathogenesis (12, 13).

Recently the clinical implications of MicroRNAs in Renal cell carcinoma have systematically been

reviewed (14) moreoverpotential roles of microRNAs as prognostic and diagnostic tools in RCC as

well as its subtypes have been reported in many studies.(4, 15-34).

Among the large number of miRs, miR-21 has received specific attention due to its relationship

with multiple cancer entities؛ it is upregulated in many tumors including breast, lung , Colon,

pancreas, prostate and stomach cancers(35-37) (38).The effect of miR-21 on carcinogenesis and

tumor progression has experimentally been analyzed(39).MiR-21 regulates epithelial-mesenchymal

transition phenotype and hypoxia-inducible factor-1a expression in breast cancer stem cell-like cells

and contributes to cell migration for metastasis(40).The global predicting role of miR-21 for

survival in patients with a variety of carcinomas is summarized in a meta-analysis (41).

To date, many researchers have published their data for the prognostic value of miR-21 in Renal

cancer, and have raised concerns about the efficiency of miR-21 as a biomarker, whereas these

issues remain questionable (4,17,19,31).

This systematic review, for the first time, will attempt to evaluate the diagnostic and prognostic

accuracy of miR-21 in RCC patients.

Objectives

Our primary objectives are to determine the diagnostic and prognostic accuracy of miR-21 in RCC

patients the secondary objectives are to determine the diagnostic and prognostic accuracy of miR-21

in RCC subtypes together with the evaluation of the heterogeneity and its potential sources in

primary studies.

Methods

Methods adopted for this systematic review have been developed in accordance with guidelines

detailed on PRISMA checklist .Besides the PRISMA Flow Diagram will be employedto describe

the flow of information through the different phases of the systematic review.(42)

The protocol of this systematic review has been registered on PROSPERO 2015 (registration

number: CRD42015025001).

Preferred Reporting Items for Systematic reviews and Meta-Analyses for Protocols 2015

(PRISMA-P 2015) have been used for preparing and reporting this protocol for the systematic

review. (43)

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Criteria for considering studies for this systematic review

Types of studies The present paper incorporates all the observational studies such as cross sectional, case control,

and cohort. Apparently studies assessing animal models, review articles, proceeding conferences

and case series studies on dead patients from RCC are excluded.

Participants

Adult patients of both sexes with diagnosis of RCC will be included. Tumor classification and stage

were diagnosed according to TNM system and 2004 WHO classification (44, 45).

Index test

miR-21

miRs are highly conserved small noncoding RNA strands, approximately 22 nucleotides in length

that post-transcriptionallyregulate gene expression and appear to be modulators of urologic cancers

(46, 47).miR-21 is one of the most frequently studied cancer-related miRNAs and dysregulated in

most cancers by acting as oncogene.(48)

RCC has considerably explored the upregulation of mir-21 in comparison with healtht kidneys (4,

39). There exists a significant difference between expression levels of miR-21and RCC subtypes

with respect to the highest levels in ccRCC and pRCC subtypes.MiR-21 levels are associated with

higher stage and grade, so its expression level can be used in as a diagnostic marker in

distinguishing RCC subtypes (4).The expression level of miR-21 is correlated with a span of 5-year

survival and pathological stage in RCC (49). Patients who are miR-21 positive have statistically

shorter disease free and overall survival rates (4)In the current study, Mir-21 detection in RCC

tissues will be defined on the basis of Quantitative Real-Time RT-PCR.

There are several steps for mir-21 detection in RCC tissues based on Quantitative Real-Time RT-

PCR.

1. Total RNA have been extracted from RCC samples (formalin-fixed, paraffin-embedded tissues or

serum) using commercial kits according to the manufacturer’s protocol.

2. RNA concentrations and A260/280 ratio with a Nano Drop have been determined.

3. The resulting miRNA for quantitative Real Time PCR (qRT-PCR) have been retained and

miRNA expression with TaqMan MicroRNA Assays have been measured.

4. Reverse transcription of miR-21 with a specified amount of total RNA by making use of TaqMan

MicroRNA specific Reverse Transcription Kit has been performed.

5. Cycling conditions for miR-21 according to manufacturer’s protocols have been chosen.

6. All reactions have been performed in triplicates.

7. Values of miR-21 in regard to a small nuclear RNA as reference gene have been normalized.

8.Relative over- or under expression of miR-21 in patients samples have been compared to the

normal ones by the ∆∆Ct method assuming that equal RNA concentrations and efficiency of qRT-

PCR have been completed.(4,7,19,31)

Gold standards

Studies will be eligible for this review if clinical diagnostic criteria recommended by WHO

classification and TNM system of the renal tumors of the adults are used as primary reference


radical nephrectomy or biopsies .Because clinical practice guidelines are routinely updated, we will

use the criteria of reference standard that the primary studies adopted.

Survival measures such as Disease Free survival (DFS), Recurrence Free, Survival (RFS) and

Hazard Ratio with 95% CI will be used as reference standards for prognostic accuracy.

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Target conditions

The target conition for diagnostic and prognostic value will be verified by RCC and survival

measures respectively.

Outcomes

The primary outcomes will be all survival measures of RCC patients and the secondary outcomes

will be to determine prognostic accuracy of mir-21 in different subtypes of renal cancer, as well as,

determination of the heterogeneity and its potential sources.


Electronic searches

We will search the following databases: PubMed, Web of Sciences, Scopus, up to 31 July

2015).The searches will be refined using the bloom term “AND” between the topics of RCC and

index test (miR-21).


MeSH tags were found in PubMed. The details of the PubMed database search strategy and syntax

aresequentially provided as below.

1. miR21

2. miR-21

3. microRNA-21

4. miRNA-21

5. hsa-mir-21

7.1 or 2 or 3 or 4 or 5

8. Renal

9. Kidney

10.8 or 9

11.7 AND 10

(miR21 [tiab] OR microRNA-21[tiab] OR miRNA-21[tiab] OR hsa-mir-21[tiab] OR miR-21[tiab])

AND (kidney [tiab] OR renal [tiab])

Other resources

Reference lists of relevant primary studies, reviews and key journals will be searched for additional

studies.


A data extraction form will be developed and study data will independently be assessed and

extracted by two reviewers, AR and RR.

The following data will be extracted from all of the included studies:

1. Study characteristics (author, year of publication, study design, setting, locations and patient


2. Test characteristics (test type, test condition, prespecified cutoff values, sampling protocol, and

criteria for test positivity)

3. Reference standards (histopathological diagnosis or criteria of the guideline used)

4. participants’characteristics (age, gender, ethnicity, disease type, tumor sizes and tumor stages)

5. Statistics for meta-analysis are: (true positives (TP), False positives (FP), true negatives (TN) and

false negatives (FN), Sensitivity, Specificity, Positive and Negative Likelihood Ratios and Area

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under the Curve(AUC) for diagnostic value and Overall survival(OS), Disease Free survival

(DFS)Recurrence Free Survival(RFS) for prognostic values.

The unavailable information of the studies will be sought from authors by email. Any discrepancies



Methodological quality of primary studies will be assessed according toa revised tool devised for

QUADAS-1 quality assessment. (50)

The defined questions will be answered as yes, no, or unclear and the score of each article will be

calculated. Two authors (A.R and R.R) will initially complete the pilot QUADAS-1 items in three

studies. If poor agreement is noted we will refine the tool then the updated tool will be applied to

complete the assessment for all included studies. Disagreements will be resolved by consensus.

Assessment of heterogeneity

To investigate heterogeneity, we will include study design (prospective or retrospective and year of

publication), population characteristics (gender, ethnicity, age, disease types, and stage

distribution), test characteristics (cutoff value, test type, and the total number of tests per screening

round), and versions of reference standards as our study-level variables. We will test these study-

level covariates in the bivariate model in the common threshold or add them to the Rutter and

Gatsonis HSROC model to evaluate heterogeneity in test threshold, diagnostic accuracy, and the

shape of curves. The likelihood ratio test will be used to determine the statistical significance of the

covariates included in the models. (51)


The publication bias will be assessed by funnel plots (ie, plots of study results against precision)

and Begg and Egger’s tests.

Statistical analysis and data synthesis Descriptive analysis:

All included studies will be overviewed and presented in two separate tables. The firsttable will

provide details on study quality according to the review-specific QUADAS-1 tool. The other table

will include study design, participants, test characteristics, results, outcomes and reference

standards.The following test characteristics will be extracted into 2 × 2 tables(TP, FP, TN, and

FN)for all included studies, Study-specific estimations of sensitivity and specificity with 95%

confidence intervals will be displayed in forest plots using Review Manager (Version 5.2). These


and brands of the index test.

Collecteddata about outcomes such as Disease Free survival (DFS), Recurrence Free Survival

(RFS) and Hazard Ratio with 95% CI will be used for prognostic value of index test.

Inferential statistics:

All the included studies will be synthesized after systemic review. Study-specific Hazard Ratio,

Sensitivity and Specificity estimates will be pooled using a fixed-effect model if no significant

heterogeneity exists, otherwise a random-effect model will be applied. The extent of heterogeneity

throughout the studies will be checked up using the chi-squared test and I-squared test; p=0.10

and/or I2>50% indicate significant heterogeneity. To evaluate sources of heterogeneity, sub-group

analysis and meta-regression will be used.

The potential for publication bias was assessed using funnel plot, the Begg rank correlation method,

and the Egger weighted regression method. A p-value of ≤ 0.05 will be considered to be statistically

significant.

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All of the mentioned analyses will be performed using STATA version 11.1 software. (Stata Corp



A sensitivity analysis will be conducted to test the impact of the results in respect to the

methodologically quality items rated by the QUADAS-1 tool criteria.

We will implement sensitivity analyses to explore the impacts of methodological quality and

sample size on the robustness of review conclusions. Meta-analyses will be repeated after excluding

studies with lower methodological quality and studies with sample sizes much bigger than those of

other studies. Sensitivity analyses will be reported with a summary table and reviewed conclusions

will be interpreted with regard to making comparisons between the two meta-analyses.

Subgroup analyses

Subgroup analysis will be conducted according to region, gender, quality, sample size and cancer

subtype.

Discussion

Despite comprehensive understanding of the biology of renal cell carcinoma and recent advances in

novel therapeutic strategies, the lack of molecular biomarkers for early detection and classification

of RCC and poor prognosis for these patients are major complications.

Mir-21 is a tumor marker that is up regulated in RCC as many tumors. In RCC Mir-21 involves in

cellular mechanisms such as growth, apoptosis, cell cycle, invasion and migration of tumor cells.

Our systematic review will clarify the prognostic and diagnostic accuracy of miR-21 in renal cell

carcinoma patients which allows patients, clinicians, and researchers to determine the diagnostic

performance of miR-21 for detection of RCC, particularly early-stage RCC, and the potential role of

this marker in prognosis of RCC patients. This systematic review will also help policy makers and

guideline developers in management of RCC patients.

Contributors MM, ZM, AR and AK were contributed to the concept and study design. AK and AR developed the

search strategies. AR and AK were contributed to implementation and analysis of the studies. AR

contributed to initial drafting, critical revision and gave approval for the manuscript to be submitted.

RR and SB revised the manuscript.AR and RR will also screen the potential studies, extract data

and assess their quality. Any discrepancies will be resolved by consensus between the two authors.

Competing interests

The authors declare no conflict of interests.

Funding

No.




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Acknowledgements

We would like to express our gratitude to Mr. Alireza Ahmadi Raad for his careful and adroit

reading and editing of the manuscript.

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References:


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14. Li M, Wang Y, Song Y, Bu R, Yin B, Fei X, et al. MicroRNAs in renal cell carcinoma: A systematic

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2014;105(11):1427-34.




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2014;35(5):4057-66.



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cancer. Oncology. 2007;72(5-6):397-402.













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51. Rutter CM, Gatsonis CA. A hierarchical regression approach to meta-analysis of diagnostic test

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Title:

Identification page 1 1a Identify the report as a protocol of a systematic review


Registration page 1 2 If registered, provide the name of the registry (such as PROSPERO) and registration number

Authors:

Contact page 1 3a Provide name, institutional affiliation, e-mail address of all protocol authors; provide physical mailing address of


Contributions page 7 3b Describe contributions of protocol authors and identify the guarantor of the review

Amendments page 1 4 If the protocol represents an amendment of a previously completed or published protocol, identify as such and list changes;


Support:

Sources page 7 5a Indicate sources of financial or other support for the review

Sponsor page 7 5b Provide name for the review funder and/or sponsor

Role of sponsor or funder

page 7

5c Describe roles of funder(s), sponsor(s), and/or institution(s), if any, in developing the protocol

INTRODUCTION

Rationale page 2 6 Describe the rationale for the review in the context of what is already known

Objectives page 1,2,3 7 Provide an explicit statement of the question(s) the review will address with reference to participants, interventions,


METHODS

Eligibility criteria page 2,3 8 Specify the study characteristics (such as PICO, study design, setting, time frame) and report characteristics (such as years


Information sources page 4 9 Describe all intended information sources (such as electronic databases, contact with study authors, trial registers or other


Search strategy page 4 10 Present draft of search strategy to be used for at least one electronic database, including planned limits, such that it could be

repeated

Study records:

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Data management page3, 4 11a Describe the mechanism(s) that will be used to manage records and data throughout the review

Selection process page 1 11b State the process that will be used for selecting studies (such as two independent reviewers) through each phase of the


Data collection process page 5 11c Describe planned method of extracting data from reports (such as piloting forms, done independently, in duplicate), any


Data items page 6 12 List and define all variables for which data will be sought (such as PICO items, funding sources), any pre-planned data


Outcomes and prioritization page 4 13 List and define all outcomes for which data will be sought, including prioritization of main and additional outcomes, with

rationale

Risk of bias in individual studies

page 6

14 Describe anticipated methods for assessing risk of bias of individual studies, including whether this will be done at the


Data synthesis page 6

page 6

page 6

page 6

15a Describe criteria under which study data will be quantitatively synthesised





Meta-bias(es) page 6 16 Specify any planned assessment of meta-bias(es) (such as publication bias across studies, selective reporting within studies)

Confidence in cumulative evidence

Page 5 17

Describe how the strength of the body of evidence will be assessed (such as GRADE)






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Journal: BMJ Open

Manuscript ID bmjopen-2015-009667.R2


Date Submitted by the Author: 26-Nov-2015

Complete List of Authors: Rasti, Arezoo; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of) Mehrazma, Mitra; Iran University of Medical Sciences (IUMS), Oncopathology Research Center; Iran University of Medical Sciences, Department of Pathology Madjd, Zahra; Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran; Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Department of Molecular Medicine

Keshtkar, Abbas; School of Public Health,Tehran University of Medical Sciences, Department of Health Sciences Education Development Roudi, Raheleh; Iran University of Medical Sciences (IUMS), Oncopathology Research Center Tehran, Iran (the Islamic Republic of) Tehran Babashah, Sadegh; Faculty of Biological Sciences, Tarbiat Modares University, Department of Molecular Genetics


Oncology

Secondary Subject Heading: Pathology, Urology, Diagnostics



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protocol


1,2, Zahra Madjd*

1,2,3, Abbas Ali Keshtkar

4, Raheleh Roudi

1,

Sadegh Babashah5


2. Department of Pathology, Iran University of Medical Sciences, Tehran, Iran 3. Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of


4. Department of Health Sciences Education Development, School of Public Health, Tehran

University of Medical Sciences

5. Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares

University, Tehran, Iran

Correspondence to

[email protected] [email protected]

Abstract

Introduction:

Renal cell carcinoma (RCC) is the most common neoplasm in adult kidneys. One of the most

important unmet medical needs in RCC is a prognostic biomarker to enable identification of

patients at high risk of relapse after nephrectomy. New biomarkers can help improve diagnosis

and hence the management of renal cancer patients. Thus, this systematic review aims to clarify

the prognostic and diagnostic accuracy of miR-21 in RCC patients.



patients with renal cancer. The index test and reference standards should ideally be performed on

all patients. We will search PubMed, SCOPUS, and ISI Web of Science with no restriction of

language. The outcome will be survival measures in adult patients with RCC.

Study selection and data extraction will be performed by two independent reviewers. QUADAS-

1 will be used to assess study quality. Publication bias and data synthesis will be assessed by

funnel plots and Begg’s and Egger’s tests using Stata software version 11.1.




International Prospective Register of Systematic Reviews, registration number

CRD42015025001.


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This systematic review, for the first time, will evaluate the prognostic and diagnostic accuracy of

miR-21 in patients with RCC through a search of several databases without placing restrictions

on language.

The study screening, data extraction, and assessing risk of bias of the current study will be


We expect some potential heterogeneities between previous studies, including stage and


Introduction

Renal cell carcinoma (RCC) is the most common neoplasm in adult kidneys (1). The incidence

of RCC is increasing by approximately 20% per annum (2). Distinguishing the RCC subtypes is

of clinical importance because they have different prognoses and subsequently require different

management plans (3). The most common form of RCC is the clear subtype RCC (ccRCC) that

accounts for 75%–80% of cases; other less common subtypes include papillary RCC (pRCC),

chromophobe RCC (chRCC), and collecting duct carcinoma (4). Although surgery is curative for

the localized disease, a significant proportion of patients relapse or metastasize. Metastatic RCC

is difficult to treat. The 5-year survival rate for metastatic RCC is dismal at less than 10% (5). At

present, there is an absence of biomarkers for the early detection and follow-up of the disease,

which is responsible for the late diagnosis and the subsequent poor prognosis (6).

Factors influencing RCC prognosis can be classified into anatomic, histological, clinical, and

molecular (2). Pathological stage, based on the size and extent of invasion by the tumor, is the

most accurate indicator of prognosis (7). Recent evidence shows that molecular signatures can

classify RCC subtypes more accurately than morphological characteristics (8–10). Micro RNAs

(miRNAs) are small noncoding RNA nucleotides that post-transcriptionally regulate the

expression of their target proteins (4). The first report suggesting a role of miRNAs in cancer

was published in 2002 (11).

miRNAs are actively involved in kidney cancer pathogenesis (12, 13). Recently, the clinical

implications of miRNAs in RCC have been systematically reviewed (14); moreover, the

potential roles of miRNAs as prognostic and diagnostic tools in RCC as well as its subtypes have

been reported in several studies (4, 15–34).

Among the numerous miRNAs, miR-21 has received specific attention because of its

relationship with multiple cancers. It is up regulated in several tumors, including breast, lung,

colon, pancreas, prostate, and stomach cancers (35–38). The effect of miR-21 on carcinogenesis

and tumor progression has been experimentally analyzed (39). miR-21 regulates epithelial–

mesenchymal transition phenotype and hypoxia-inducible factor-1α expression in breast cancer

stem cell-like cells and contributes to cell migration in metastasis (40). The global prediction role

of miR-21 for survival in patients with various carcinomas has been summarized in a meta-

analysis (41).

Till date, several researchers have published their data on the prognostic value of miR-21 in RCC

and have raised concerns about its prognostic accuracy as a biomarker; however, these issues

remain questionable (4, 17, 19, 31). This systematic review aims to evaluate the diagnostic and

prognostic accuracy of miR-21 in RCC patients.

Objectives

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The primary objectives are to determine the diagnostic and prognostic accuracy of miR-21 in

RCC patients. The secondary objectives include determining the diagnostic and prognostic

accuracy of miR-21 in RCC subtypes together with the evaluation of heterogeneity and its

potential sources in primary studies.

Methods

The methods adopted for this systematic review have been developed in accordance with the

guidelines detailed on the PRISMA checklist. In addition, the PRISMA Flow Diagram will be

employed to describe the flow of information through the different phases of this systematic

review (42). The protocol of this systematic review has been registered in PROSPERO 2015

(registration number: CRD42015025001). The Preferred Reporting Items for Systematic reviews

and Meta-Analyses for Protocols 2015 (PRISMA-P 2015) have been used for preparing and

reporting the protocol of this systematic review (43).

Inclusion criteria

Types of studies Observational studies (cross-sectional, case-control, and cohort) will be included. Studies

assessing animal models, review articles, proceedings, conferences, and case series studies on

dead patients from RCC will be excluded.

Participants

Adult patients of both sexes with a diagnosis of RCC will be included. Tumor classification and

stage will be diagnosed according to the TNM system and 2004 WHO classification (44,45).

Index test

miR-21

miRNAs are highly conserved small noncoding RNA strands, approximately 22 nucleotides in

length that post-transcriptionally regulate gene expression and appear to be modulators of

urological cancers (46, 47). miR-21 is one of the most frequently studied cancer-related miRNAs

and is dysregulated in most cancers by acting as an oncogene (48).

RCC considerably exploits the up regulation of miR-21 in comparison with healthy kidneys (4,

39). A significant difference exists between the expression levels of miR-21and RCC subtypes

with respect to the highest levels in ccRCC and pRCC subtypes. miR-21 levels are associated

with higher stage and grade, and hence its expression level can be used as a diagnostic marker in

distinguishing RCC subtypes (4). The expression level of miR-21 is correlated with a span of 5-

year survival and pathological stage in RCC (49). Patients who are miR-21 positive have

statistically shorter disease-free survival (DFS) and overall survival (OS) rates (4). In the current

review, miR-21 detection in RCC tissues will be defined on the basis of quantitative real-time

polymerase chain reaction (qRT-PCR).

There are several steps for miR-21 detection in RCC tissues based on qRT-PCR, which are as

follows:

1. Total RNA is extracted from RCC samples (formalin-fixed, paraffin-embedded tissues or

serum) using commercial kits, according to the manufacturer’s protocol.

2. RNA concentrations and A260/280 ratio with a Nano Drop are determined.

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3. The resulting miRNA for qRT-PCR is retained, and miRNA expression is measured by

TaqMan miRNA assays.

4. Reverse transcription of miR-21 with a specified amount of total RNA using the TaqMan

MicroRNA-specific Reverse Transcription Kit is performed.

5. The cycling conditions for miR-21 are selected, according to the manufacturer’s protocols.

6. All reactions are performed in triplicates.

7. The values of miR-21 with respect to a small nuclear RNA as a reference gene are normalized.

8. The relative over- or under-expression of miR-21 in patient samples is compared to that in

normal subjects by the ∆∆Ct method (4, 7, 19, 31).

Gold standards

Studies will be eligible for this review if the clinical diagnostic criteria recommended by the

WHO classification and TNM system of the renal tumors of adults are used as primary reference


radical nephrectomy or biopsies. Because clinical practice guidelines are routinely updated, we

will use the criteria of reference standard that the primary studies adopted. Survival measures

such as DFS, OS, recurrence-free survival (RFS), and hazard ratio with 95% confidence interval

(CI) will be used as reference standards for prognostic accuracy.

Target conditions

The target conditions for diagnostic and prognostic values will be verified by RCC diagnosis and

survival measures, respectively.

Outcomes

The primary outcomes will be all survival measures of RCC patients. The secondary outcomes

will include the determination of prognostic accuracy of miR-21 in different subtypes of renal

cancer, as well as the determination of heterogeneity and its potential sources.


Electronic searches

We will search the following databases: PubMed, Web of Science, and Scopus, till July 31,

2015.


MeSH tags were found in PubMed. The details of the PubMed database search strategy and

syntax are sequentially provided below.

1. miR21

2. miR-21

3. microRNA-21

4. miRNA-21

5. hsa-mir-21

6. 1 or 2 or 3 or 4 or 5

7. Renal

8. Kidney

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9. 7 or 8

10. 6 and 9

[miR21 (tiab) OR microRNA-21(tiab) OR miRNA-21(tiab) OR hsa-mir-21(tiab) OR miR-

21(tiab)] AND [kidney (tiab) OR renal (tiab)]

Other resources

Reference lists of relevant primary studies, reviews, and key journals will be searched for

additional studies.


A data extraction form will be developed, and study data will be independently assessed and

extracted by two reviewers, AR and RR.

The following data will be extracted from all the included studies:

1. Study characteristics (author, year of publication, study design, setting, locations, and patient


2. Test characteristics (test type, test condition, pre-specified cutoff values, sampling protocol,

and criteria for test positivity).

3. Reference standards (histopathological diagnosis or criteria of the guideline used).

4. Participants’ characteristics (age, gender, ethnicity, disease type, tumor sizes, and tumor

stages).

5. Statistics for meta-analysis, including true positives (TP), false positives (FP), true negatives

(TN), false negatives (FN), sensitivity, specificity, positive and negative likelihood ratios, area

under the curve (AUC) for diagnostic value, OS, DFS, and RFS for prognostic values.

Unavailable information from included studies will be sought from authors by email. Any

discrepancies will be resolved by consensus between AR and RR.


The methodological quality of primary studies will be assessed by a revised tool devised for

QUADAS-1 quality assessment (50). The defined questions will be answered as yes, no, or

unclear, and the score of each article will be calculated. Two authors (AR and RR) will initially

complete the pilot QUADAS-1 items in three studies. If the authors do not agree, we will refine

the tool, and then the updated tool will be applied to complete the assessment for all included

studies. Disagreements will be resolved by consensus.

Assessment of heterogeneity

To investigate heterogeneity, we will include the study design (prospective or retrospective and

year of publication), population characteristics (gender, ethnicity, age, disease types, and stage

distribution), test characteristics (cutoff value, test type, and the total number of tests per

screening round), and versions of reference standards as our study-level variables. We will test

these study-level covariates in the bivariate model in the common threshold or add them to the

Rutter and Gatsonis HSROC model to evaluate the heterogeneity in the test threshold, diagnostic

accuracy, and the shape of curves. The likelihood ratio test will be used to determine the

statistical significance of the covariates included in the models (51).

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The publication bias will be assessed by funnel plots (i.e., plots of study results against precision)

and Begg’s and Egger’s tests.

Statistical analysis and data synthesis Descriptive analysis

All included studies will be overviewed and presented in two separate tables. The first table will

provide details on the study quality according to the review-specific QUADAS-1 tool. The other

table will include the study design, participants, test characteristics, results, outcomes, and

reference standards. The following test characteristics will be extracted into 2 × 2 tables (TP, FP,

TN, and FN) for all included studies. Study-specific estimations of sensitivity and specificity

with 95% CI will be displayed in forest plots using Review Manager (version 5.2). These


and brands of the index test. The collected data about the outcomes such as DFS, OS, RFS, and

hazard ratio with 95% CI will be used for calculating the prognostic value of the index test.

Inferential statistics

All the included studies will be synthesized after a systemic review. Study-specific hazard ratio,

sensitivity, and specificity estimates will be pooled using a fixed effect model, if no significant

heterogeneity exists; otherwise, a random effect model will be applied. The extent of

heterogeneity throughout the studies will be checked using the chi-squared test and I-squared

test; p = 0.10 and/or I2 > 50%, indicating significant heterogeneity. To evaluate the sources of

heterogeneity, subgroup analysis and meta-regression will be performed.

The potential for publication bias will be assessed using funnel plot, Begg’s rank correlation

method, and Egger’s weighted regression method. A p value of ≤0.05 will be considered to be

statistically significant.

All the mentioned analyses will be performed using STATA version 11.1 software (StataCorp



A sensitivity analysis will be conducted to test the impact of the results with respect to the

methodologically quality items rated by the QUADAS-1 tool criteria. We will also implement

sensitivity analyses to explore the impacts of methodological quality and sample size on the

robustness of review conclusions. Meta-analyses will be repeated after excluding studies with

lower methodological quality and studies with sample sizes much larger than those of other

studies. Sensitivity analyses will be reported with a summary table, and reviewed conclusions

will be interpreted by making comparisons between the two meta-analyses.

Subgroup analyses

Subgroup analyses will be conducted according to the region, gender, quality, sample size, and

cancer subtype.

Discussion

Despite the comprehensive understanding of the biology of RCC and recent advances in novel

therapeutic strategies, the lack of molecular biomarkers for the early detection and classification

of RCC and the poor prognosis for these patients are major complications. miR-21 is a tumor

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marker that is up regulated in RCC and in several tumors. In RCC, miR-21 is involved in cellular

mechanisms such as growth, apoptosis, cell cycle, invasion, and migration of tumor cells.

Our systematic review will clarify the prognostic and diagnostic accuracy of miR-21 in RCC

patients, which would allow patients, clinicians, and researchers to determine the diagnostic

performance of miR-21 for the detection of RCC, particularly early stage RCC, and the potential

role of this marker in the prognosis of RCC patients. This systematic review will also help

policymakers and guideline developers in the management of RCC patients.

Contributors MM, ZM, AR, and AK contributed to the concept and study design. AK and AR developed the

search strategies. AR and AK contributed to the implementation and analysis of the studies. AR

contributed to initial drafting and critical revision and approved the manuscript for submission.

RR and SB revised the manuscript. AR and RR will also screen potential studies, extract data,

and assess their quality. Any discrepancies will be resolved by consensus between the two

authors.

Competing interests

The authors declare no conflict of interests.

Funding

No.


All recorded data from the data extraction process will be available on request to the extent that

it is not included in the systematic review article.

Acknowledgments

We would like to acknowledge the efforts of Mr. Rahul Krishan Arora and Mr. Alireza Ahmadi

Raad for their diligent and thoughtful editing of the manuscript.

References:


2009;115(S10):2273-81.





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Diagnostics. 2012;14(4):385-92.

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1996;335(12):865-75.













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2010;183(2):743-51.



2010;391(4):411-23.

14. Li M, Wang Y, Song Y, Bu R, Yin B, Fei X, et al. MicroRNAs in renal cell carcinoma: A systematic

review of clinical implications (Review). Oncology reports. 2015;33(4):1571-8.





2014;105(11):1427-34.




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2014;15(3):329-41.




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2013;109(10):2646-53.



Neoplasma. 2012;60(5):511-5.



9.



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pathology. 2015;185(3):693-703.






pathology. 2014;7(6):3318.



oncology. 2013;1(2):235-40.






cancer. Oncology. 2007;72(5-6):397-402.











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2011;41(11):1245-53.






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51. Rutter CM, Gatsonis CA. A hierarchical regression approach to meta-analysis of diagnostic test

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Title:

Identification page 1 1a Identify the report as a protocol of a systematic review


Registration page 1 2 If registered, provide the name of the registry (such as PROSPERO) and registration number

Authors:

Contact page 1 3a Provide name, institutional affiliation, e-mail address of all protocol authors; provide physical mailing address of


Contributions page 7 3b Describe contributions of protocol authors and identify the guarantor of the review

Amendments page 1 4 If the protocol represents an amendment of a previously completed or published protocol, identify as such and list changes;


Support:

Sources page 7 5a Indicate sources of financial or other support for the review

Sponsor page 7 5b Provide name for the review funder and/or sponsor

Role of sponsor or funder

page 7

5c Describe roles of funder(s), sponsor(s), and/or institution(s), if any, in developing the protocol

INTRODUCTION

Rationale page 2 6 Describe the rationale for the review in the context of what is already known

Objectives page 1,2,3 7 Provide an explicit statement of the question(s) the review will address with reference to participants, interventions,


METHODS

Eligibility criteria page 2,3 8 Specify the study characteristics (such as PICO, study design, setting, time frame) and report characteristics (such as years


Information sources page 4 9 Describe all intended information sources (such as electronic databases, contact with study authors, trial registers or other


Search strategy page 4 10 Present draft of search strategy to be used for at least one electronic database, including planned limits, such that it could be

repeated

Study records:

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Data management page3, 4 11a Describe the mechanism(s) that will be used to manage records and data throughout the review

Selection process page 1 11b State the process that will be used for selecting studies (such as two independent reviewers) through each phase of the


Data collection process page 5 11c Describe planned method of extracting data from reports (such as piloting forms, done independently, in duplicate), any


Data items page 6 12 List and define all variables for which data will be sought (such as PICO items, funding sources), any pre-planned data


Outcomes and prioritization page 4 13 List and define all outcomes for which data will be sought, including prioritization of main and additional outcomes, with

rationale

Risk of bias in individual studies

page 6

14 Describe anticipated methods for assessing risk of bias of individual studies, including whether this will be done at the


Data synthesis page 6

page 6

page 6

page 6

15a Describe criteria under which study data will be quantitatively synthesised





Meta-bias(es) page 6 16 Specify any planned assessment of meta-bias(es) (such as publication bias across studies, selective reporting within studies)

Confidence in cumulative evidence

Page 5 17

Describe how the strength of the body of evidence will be assessed (such as GRADE)






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Documents

BMJ Open · Hemmat Street (Highway), Next to Milad Tower, Tehran, Iran Post Code: 14496-14530 Email address: [email protected] Tel: +982186703212 Fax: +982188622608 Abstract Introduction: