Liquid biopsy in ovarian cancer: the potential of ...

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REVIEW ARTICLELiquid biopsy in ovarian cancer: the potentialof circulating miRNAs and exosomes

D193X XLYDIA GIANNOPOULOUD194X X, D195X XMARTHA ZAVRIDOUD196X X, D197X XSABINE KASIMIR-BAUERD198X X, and D199X XEVI S. LIANIDOUD200X X

ATHENS, GREECE; and ESSEN, GERMANY

From the Department of Chemistr

Cells lab, Lab of Analytical Chem

versity Campus, Athens, Greece;

Obstetrics, University Hospital of

Essen, Essen, Germany.

Submitted for Publication July 31,

8, 2018; accepted for publication O

Reprint requests: Evi S. Lianid

Analysis of Circulating Tumour C

cal Chemistry, University of A

e-mail: [email protected].

1931-5244/$ - see front matter

� 2018 Elsevier Inc. All rights rese

https://doi.org/10.1016/j.trsl.2018.1

Ovarian cancer still remains the most lethal female cancer, since in most cases it isdiagnosed at an advanced stage. Usually after completion of primary treatmentchemoD201X Xresistance occurs, and recurrent disease is finally observed. Liquid biopsy,based on minimally invasive and serial blood tests, has the advantage of followingtumor evolution in real time, offering novel insights on precision medicine. Circulat-ing tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating cell-free micro-RNAs (cfmiRNAs) and circulating exosomes represent the major components ofliquid biopsy analysis. Liquid biopsy has been already implemented in ovarian can-cer, and most studies so far are mainly focused on CTCs and ctDNA. This review ismainly focused on the clinical potential of circulating miRNAs and exosomes as asource of liquid biopsy biomarkers in ovarian cancer diagnosis, prognosis, andresponse to treatment. (Translational Research 2018; 000:1�15)

Abbreviations: AUC=area under curve; cfDNA =cell-free DNA; cfmiRNAs = cell-free micro-RNAs; CNV =copy number variations; CTCs = circulating tumor cells; ctDNA=circulating tumorDNA; ddPCR= droplet digital PCR; EMT = epithelial-to-mesenchymal transition; EVs = extracel-lular vesicles; HGSC= high-grade serous ovarian cancer; LOH = loss of heterozygosity;NGS = next-generation sequencing; OS = overall survival; PARP = poly ADP-ribose polymerase;PFS = progression-free survival; ROC= receiver operating characteristic; snRNA= small nuclearRNA

INTRODUCTION

Ovarian cancer is the most lethal among gynecologi-

cal malignancies and represents the fifth cause of can-

cer-related mortality in women. D202X X1 D203X X Epithelial ovarian

y, Analysis of Circulating Tumor

istry, University of Athens, Uni-

Department of Gynecology and

Essen, University of Duisburg-

2018; received submitted October

ctober 8, 2018.

ou, Department of Chemistry,

ells lab, Laboratory of Analyti-

thens, 15771, Athens, Greece.

rved.

0.003

cancer is the main type, characterized by remarkable

histological and molecular heterogeneity. In the major-

ity of new incidents, the disease is diagnosed at an

advanced stage and regardless of the initial response to

treatment, chemo D204X Xresistance is observed. D205X X2, D206X X3 D207X XPrimary epi-

thelial ovarian cancer is treated with surgical removal

of the tumor and a subsequent combination of platinum

and taxane-based adjuvant chemotherapy. D208X X4 D209X XThe newest

therapeutic approaches include Bevacizumab and the

poly ADP-ribose polymerase (PARP) inhibitor Ola-

parib; however, these targeted therapy agents are

applied only in certain clinical situations. D210X X5, D211X X6D212X X

Metastasis in ovarian cancer is unique among all

solid malignancies. It occurs via 2 D213X Xdistinct routes: the

transcoelomic passive dissemination of tumor cell

spheroids in the peritoneal cavity through ascites and

the hematogenous metastasis of ovarian cancer cells in

systemic circulation followed by the preferred seeding

1

[email protected]

https://doi.org/10.1016/j.trsl.2018.10.003

ARTICLE IN PRESSTranslational Research

2 Giannopoulou et al 2018

to the omentum. D214X X7, D215X X8D216X XThe final stage of both metastatic

routes is the colonization of cancer cells originating

from the ovary to distal organs, mainly the liver, the

spleen, and the lungs. D217X X9 D218X X

Liquid biopsy is a minimally invasive blood-based

approach that has the potential of providing informa-

tion on prognosis, response to therapeutic regimens

and recently even on early diagnosis and population

screening. D219X X10, D220X X11 D221X XThe detection and molecular characteri-

zation of circulating tumor cells (CTCs), the detection

of circulating tumor DNA (ctDNA), circulating cell-

free microRNAs (cfmiRNAs), and circulating extracel-

lular vesicles D222X Xlike exosomes, in the peripheral blood of

cancer patients, represent the main liquid biopsy

approaches (FigD223X X 1).11�14 It has also been recently

found that tumor-educated platelets can transfer

nucleic acids from the tumor, mainly RNA, to distant

organs. D224X X15 D225X XThe clinical significance of CTCs and ctDNA

in ovarian cancer has been intensively investigated in a

large number of research studies during the last 2 D226X Xdeca-

des and latest developments in this field have been

recently reviewed. D227X X16�21D228X X

Circulating miRNAs are a subclass of small nonD229X Xcoding

RNAs that have been detected in almost all body fluids.

Circulating miRNAs are involved in cell-to-cell commu-

nication and their main role is the up- or down-regulation

of the expression of their target genes. miRNAs biogene-

sis occurs first in the nucleus and then in the cytoplasm;

afterwards, they are released in the circulation as cell-

free miRNAs bound in specific RNA-binding proteins

(circulating cfmiRNAs) or encapsulated in exosomes

Fig. 1. Liquid biopsy in

(circulating exosomal miRNAs) along with proteins, lip-

ids, and nucleic acids.D230X X14,D231X X22,D232X X23D233X XExosomes are a subcategory

of extracellular vesiclesD234X X secreted by living cells in the

extracellular space or in blood circulation; in cancer

patients, exosomes are also secreted by tumor cells and it

is now believed that they play important roles in the met-

astatic process.D235X X24�26D236X XIn ovarian cancer, a large number of

studies suggest a possible role of exosomes in early diag-

nosis, prognosis, and response to treatment.D237X X26�28D238X X

In this review, we provide a brief description of the

most recent studies on CTCs and ctDNA in ovarian

cancer and a detailed overview of studies on circulating

cfmiRNAs and exosomes in ovarian cancer. We also

focus on the clinical significance of liquid biopsy in

ovarian cancer and attempt to clarify their role in early

diagnosis, prognosis, and response to treatment.

CIRCULATING TUMOR CELLS D239X X

In epithelial ovarian cancer, CTCs are involved in

the hematogenous metastatic routeD240X X17, D241X X29 D242X X Up to now,

many studies attempted to evaluate the clinical signifi-

cance of CTCs, especially their role as prognostic and/

or predictive biomarkers. D243X X19, D244X X20 D245X XA variety of technologies

have been used for CTCs enrichment and detection in

ovarian cancer; CTCs isolation and/or enrichment is

based mainly on immunomagnetic particles or is size-

based, while CTCs detection is based on immunocyto-

chemistry approaches (microscopic detection or the

FDA-cleared CellSearch D246X X system, Menarini, Silicon

Biosystems), reverse transcription polymerase chain

ovarian cancer.


ARTICLE IN PRESSTranslational ResearchVolume 00 Giannopoulou et al 3

reaction (RT-PCR) (AdnaTest, QIAGEN, Hilden, Ger-

many) and RT-qPCR. D247X X17, D248X X20 D249X X Novel technologies for

CTCs isolation and enrichment based on their physical

properties were also introduced by several research

groups, such as microfluidic devices for the isolation

and detection of CTCs in ovarian cancer patients. D250X X30, D251X X31 D252X X

The microfluidics-based ParsortixD253X X technology (Angle

plc) has been recently used for the D254X XCTC enrichment,

followed by RT-qPCR for CTC detection and molecu-

lar characterization. D255X X31 D256X X

A recent study attempted to investigate the incidence

of epithelial-to-mesenchymal transition (EMT)-like

CTCs in ovarian cancer patients, prior to surgery and

after platinum-based chemotherapy at the gene expres-

sion level. D257X X32 D258X XThe EMT-associated transcripts analyzed

were PIK3CA, AKT-2, and TWIST. Before surgery, the

presence of PI3K+EMT-like CTCs in combination

with epithelial CTCs was significantly associated with

worse overall survival (OS). In the International Feder-

ation of Gynecology and Obstetrics (FIGO) stage I-III

patients with residual tumor burden after surgery,

EMT-like CTCs were observed; furthermore, epithelial

CTCs significantly correlated with shorter progression-

free survival (PFS) and OS and the presence of PI3K+

CTCs was correlated with decreased OS. D259X X32 D260X X

In a recent study performed with a large group of

patients with ovarian cancer, which was conducted to

investigate whether the presence of CTCs could reveal

minimal residual disease, univariate, and multivariate

analysis indicated a worse OS in CTC positive patients

at the time of diagnosis. D261X X33 D262X X The alterations between

CTCs and the immune system were also investigated,

in a very recent study from the same group. D263X X34 D264X XThe pres-

ence of the immune activation marker neopterin and

the ratio of kynurenine to tryptophan were evaluated in

matched plasma samples from ovarian cancer patients,

where CTCs have already been detected. D265X X35 D266X XAt the time

of diagnosis and at follow-up, both, neopterin and

kynurenine to tryptophan levels were significantly

increased in PPIC positive CTCs when compared to

benign disease, suggesting that immune responses

occur not only in the primary tumor but also in the

circulation. D267X X34 D268X X

The potential of CTCs detection for the diagnosis of

ovarian cancer was evaluated in a recent study where a

size-based microfluidic device was used for CTCs isola-

tion, followed by their detection and identification

through immunofluorescent staining of CD45, HE4, and

the EMT markers EpCAM, pan-cytokeratins, and

vimentin. A group of women having an ovarian mass

participated in the study, and almost half of them were

diagnosed with ovarian cancer. More specifically, the

DAPI+/E&M+/CD45¡/HE4+CTC subpopulation was

significantly higher in ovarian cancer patients compared

to those with benign tumors. Furthermore, a better diag-

nostic sensitivity was estimated using receiver operating

characteristic (ROC) curves, when compared to the

most established tumor marker in ovarian cancer, serum

CA-125. The above CTC count was proposed as a bio-

marker for ovarian cancer early diagnosis in cases where

a pelvic mass was observed.D269X X36D270X XThe predictive value of

CTCs was also investigated in a very small group of

ovarian cancer patients (n = 13) that received standard

taxol and/or carboplatin chemotherapy; a statistically

significant association was observed between CTC lev-

els and disease progression.D271X X37D272X X

CIRCULATING TUMOR DNA D273X X

A number of studies on ctDNA in patients with ovar-

ian cancer attempted to evaluate its clinical value, in the

terms of early diagnosis, prognosis, and response to treat-

ment or even screening in the general population.D274X X16,D275X X38D276X XTo

this end, total ctDNA and/or circulating cell-free mito-

chondrial DNA quantification, or detection of different

genetic and epigenetic alterations, such as chromosomal

abnormalities and specific tumor loss of heterozygosityD277X X,

somatic and germline gene mutations and aberrant DNA

methylation, were correlated with diagnosis, prognosis,

and response to treatment.D278X X18D279X XIn a very recent study, short

ALU-115 repeat and long ALU-219 fragments were

amplified and used for the detection of plasma cell-free

DNA (cfDNA) concentrations and ALU-219/ALU-115

integrity index was measured in ovarian cancer patients.

Significantly higher ALU-219 fragment levels and integ-

rity index were observed in ovarian cancer when com-

pared to benign disease and healthy individuals,

suggesting a possible role of ctDNA analysis in early

diagnosis of ovarian cancer.D280X X39D281X X

Epigenetic alterations in ctDNA are also important

in ovarian cancer. It was recently shown that the meth-

ylation status of 3 D282X X genes (COL23A1, C2CD4D, and

WNT6), discovered using targeted ultra-high coverage

bisulfite sequencing, could discriminate successfully

the responders and non D283X Xresponders to platinum-based

neoadjuvant chemotherapy. D284X X40 D285X X We recently evaluated

for the first time ESR1 methylation status in plasma

ctDNA and paired primary tumors of patients with

high-grade serous ovarian cancer (HGSC), and found

an agreement between ESR1 methylation in primary

tumors and paired ctDNA. D286X X41 D287X X

Droplet digital PCR (ddPCR) was used for the eval-

uation of somatic TP53 mutations in plasma ctDNA

samples of patients with serous ovarian cancer. D288X X42, D289X X43 D290X X

TP53 mutations were investigated in serial ctDNA

samples of HGSC patients; the presence of TP53

mutant allele fractions in ctDNA, when compared to




serum CA-125, could indicate much earlier response to

chemotherapy. D291X X43D292X X

The importance of the reversion of BRCA1/2 muta-

tions in ctDNA as an indicator of response to platinum-

based and PARP inhibitor-based chemotherapy was

investigated. D293X X44, D294X X45 D295X XMore specifically, in ovarian cancer

patients with recurrent HGSC, the same BRCA1/2

reversions that were identified in paired tumor and

plasma ctDNA samples were present in the tumor.

Interestingly, resistance to platinum-based and PARP

inhibitor-based chemotherapy was observed in all

patients with BRCA1/2 reversions in ctDNA. D296X X44 D297X X The

BRCA1/2 reversion mutation status was also assessed

by using targeted massively parallel sequencing in

ctDNA samples of platinum resistant and/or refractory

ovarian cancer patients; putative BRCA1 or BRCA2

somatic reversion mutations or intragenic deletions

were detected in 21% of ovarian cancer ctDNA sam-

ples, and these results were further confirmed using

ddPCR. D298X X45 D299X X Germline and somatic BRCA1/2 mutations

were detected in plasma ctDNA of ovarian cancer

patients, by using next-generation sequencing (NGS)

technology. All germline BRCA1/2 mutations detected

in ctDNA were present in the primary tumor but

this concordance was not observed for somatic muta-

tions, indicating the incidence of intratumoral hetero-

geneity. D300X X46 D301X X

The clinical value of plasma ctDNA in treatment

monitoring was recently investigated in cases where

neoadjuvant chemotherapy is necessary. In this study,

NGS was performed in ctDNA samples before and

after neoadjuvant chemotherapy, using a specific panel

of 50 genes; before treatment, 59 genetic variants from

19 of these genes were identified but only 6 D302X X of them

were detected after neoadjuvant chemotherapy. D303X X47D304X X A

number of somatic mutations, polymorphisms, and

copy number variations D305X Xwere also detected in plasma

ctDNA samples of recurrent and drug-resistant recur-

rent ovarian cancer patients, using NGS. D306X X48 D307X X

The development of a novel massive parallel

sequencing approach named targeted error correction

sequencing, led to the identification of cancer-related

genomic changes in early stage malignancies, includ-

ing ovarian cancer. Somatic mutations of driver genes

were detected in 68% of ovarian cancer patients with

early stage disease and for all cancer types analyzed, a

high concordance between ctDNA and primary tumor

was observed. D308X X49 D309X X

The recently developed CancerSEEK blood test has

the potential to evaluate the levels of 8D310X Xproteins and the

presence of somatic mutations in 2D311X X001 genomic posi-

tions simultaneously.D312X X10D313X XWhen this test was evaluated in

1D314X X005 patients with already detected solid cancers,

including 54 patients with ovarian cancer, its sensitivity

varied in the different types of cancer tested, but the

higher diagnostic sensitivity was observed in ovarian

cancer (98%). The concordance between tissue and

ctDNA was also estimated, in cases where the primary

tumor was available, with the highest 100% concor-

dance being observed in ovarian cancer. This test could

also determine the location of the primary tumor based

on the obtained information from ctDNA and circulating

proteins. In ovarian cancer, 79% of the cases were pre-

dicted correctly as the most likely type and the percent-

age reached 92% when the second more likely type was

included.D315X X10D316X X

CIRCULATING CELL-FREE AND EXOSOMAL miRNAs

Many research studies aimed to elucidate the clinical

value of circulating cfmiRNAs and exosomal miRNAs

in serum and/or plasma of ovarian cancer patients. D317X X50�52D318X X

In a recent meta-analysis that was performed to estimate

the diagnostic significance of circulating cfmiRNAs in

ovarian cancer, the diagnostic value of circulating

cfmiRNAs was considered as moderate. However, only

ten research studies were included in the statistical ana-

lysis.D319X X53D320X XAdditionally, another nonD321X Xcoding RNA, circulat-

ing U2 small nuclear RNA (snRNA) was recently

proposed on having an important role in ovarian cancer

diagnosis.D322X X54D323X X

Most studies that investigated the clinical value of

circulating cfmiRNAs and exosomal miRNAs in ovar-

ian cancer are presented in detail below and are sum-

marized in Tables I and II. The assessment of clinical

significance resulting from these studies is also

reported (Table III). Only studies that included more

than 25 ovarian cancer patients were added in these

tables. As can be seen in Tables I�III, the results

obtained from the studies on circulating cfmiRNAs in

ovarian cancer were often controversial; a possible

explanation could be the different procedures used for

the isolation and characterization of these circulating

cfmiRNAs or the distinct reference miRNAs used for

the quantification of their expression levels.

CIRCULATING cfmiRNAs

Diagnosis. The first study that focused on circulating

cfmiRNAs in ovarian cancer was based on real-time PCR

quantification of specific miRNAs in serum samples

of ovarian cancer patients and healthy individuals;

miR-142-3p was used as a reference miRNA.D324X X55D325X XAccording

to this study, the expression of 8D326X XmiRNAs was signifi-

cantly differentiated in ovarian cancer patients when

compared to healthy controls, indicating a diagnostic

value; 5D327X X specific miRNAs were overD328X Xexpressed (miR-21,


Table I. Diagnostic value of circulating cell-free and exosomal miRNAs in ovarian cancerD1X X

OverD2X XexpressedmiRNAs

UnderD3X XexpressedmiRNAs

Biologicalspecimen

OvarianD4X Xcancerpatients

OvarianD5X Xcancersubtype

Healthycontrols

Detectiontechnology

ReferencemiRNA

Reference Year

- miR-193a-5p Serum 45 serous: 22,mucinous: 15,endome-trioid: 8

healthy con-trols: 40benign cases:30

RT-qPCR U6 snRNA Ren et al. D6X X85D7X X 2018

miR-21 - Serum 60 serous: 31,mucinous:13,other sub-types: 16

30 RT-qPCR U6 snRNA Mahmoud etal. D8X X62D9X X

2018

miR-375, miR-210, miR-150-5p, miR-181-5p

- Serum 56 HGSC 30 RT-qPCR UniSp3template

Shah et al.D10X X87D11X X 2018

Differentiallyincreased(neuralnetwork)

Differentiallydecreased(neural network)

Serum 168 serous: 100,endometrioidand clearcell: 48,mucinous: 10


RT-qPCR miR-423-3p,miR-103a-3p

Elias et al. D12X X86D13X X 2017

miR-142-3p,miR-26a-5p,let-7d-5p,miR-374a-5p,miR-766-3p,miR-200a-3p,miR-130b-3p,miR-328-3p

- Serum 155 serous: 112,clear cell: 19,endome-trioid: 13,mucinous: 11


RT-qPCR andNGS

miR-16 Yokoi et al.D14X X83 D15X X 2017

miR-1246, miR-595, miR-2278

- Serum 168 HGSC 65 RT-qPCR miR-15b Todeschini etal. D16X X82D17X X

2017

miR-376a - Serum 32 EOC, no spe-cific subtype

10 RT-qPCR U6 snRNA Yang et al. D18X X81D19X X 2017

miR-125b - Serum 135 HGSC: 89, clearcell: 24,endome-trioid: 12,other sub-types: 10

benign cases:54

RT-qPCR miR-16 Zhu et al.D20X X78 D21X X 2017

- miR-148a Plasma 102 EOC, no spe-cific subtype

70 RT-qPCR U6 snRNA Gong et al. D22X X80D23X X 2016

miR-373, miR-200a, miR-

- Serum 60 serous: 43,other

healthy con-trols: 32

RT-qPCR miR-484 Meng et al.D24X X79 D25X X 2016

(continued on next page)

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Table I. (Continued)



Biologicalspecimen



Healthycontrols

Detectiontechnology

ReferencemiRNA

Reference Year

200b, miR-200c

subtypes: 2,unknown: 15

benign cases:20

miR-125b - Serum 70 mucinous: 36,serous: 17,other sub-types: 17

70 RT-qPCR U6 snRNA Zuberi et al. D26X X77D27X X 2016

- miR-199a Serum 70 mucinous: 36,serous: 17,other sub-types: 17


miR-373, miR-200a, miR-200b, miR-200c

- Serumexosomes

163 serous: 120,other sub-types: 15,unknown: 28


RT-qPCR miR-484 Meng et al.D30X X94 D31X X 2016

miR-7, miR-429 miR-25, miR-93 Serum 180 EOC, no spe-cific subtype

66 RT-qPCR miR-484 Meng et al.D32X X75D33X X 2015

miR-200b - Plasma 51 EOC, no spe-cific subtype


RT-qPCR miR-191 Kapetanakis etal. D34X X59D35X X

2015

miR-200a, miR-200b, miR-200c

- Serum 70 mucinous: 36,serous: 17,other sub-types: 17


- miR-145 Serum 84 endometrioid:24, serous: 18,clear cell: 17,mucinous: 12,mixed: 13


RT-qPCR U6 snRNA Liang et al. D38X X66D39X X 2015

miR-200c, miR-141

- Serum 74 serous: 16,endome-trioid: 15,clear cell: 14,mucinous: 12,undifferenti-ated: 17

50 RT-qPCR NR Gao et al.D40X X74 D41X X 2015

- let-7i-5p, miR-122,miR-152-5p, miR-25-3p

Serum 25 Serous benign cases:20

RT-qPCR has-miR-103a-3p, miR-27b-3p, miR-30b-

Langhe et al. D42X X71D43X X 2015


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Table I. (Continued)



Biologicalspecimen



Healthycontrols

Detectiontechnology

ReferencemiRNA

Reference Year

5p, miR-101-3p

miR-26a - Plasma 26 serous: 15,other sub-types: 11

19 RT-qPCR cel-miR-39 Shen et al.D44X X64D45X X 2014

miR-22, miR-93,miR-451

miR-106b Serum 31 EOC, no spe-cific subtype

healthy con-trols: 8 benigncases: 23

RT-qPCR andNGS

miR-16 Ji et al.D46X X73D47X X 2014

miR-625-3p,miR-720, miR-1274a

19 miRs including:miR-19b, miR-223,miR-16, miR-150,miR-20a, miR-126,miR-1290

Plasma 42 Serous healthy con-trols: 23benign cases:36

RT-qPCR Presurgicalsamples: miR-320, miR-720,miR- 1274b,U6 snRNA Allother sam-ples: 14miRNAs

Shapira et al.D48X X72 D49X X 2014

miR-205 let-7f Plasma 360 serous: 179,endome-trioid: 86,adenocarci-noma: 47,mucinous: 33,clear cell: 15

200 RT-qPCR NR Zheng et al.D50X X67D51X X 2013

miR-21 - Serum 94 serous: 68,other sub-types: 26

40 RT-qPCR U6 snRNA Xu et al. D52X X63D53X X 2013

miR-16, miR-21,miR-191, miR-4284

- Plasma 35 Endometriosis-associated(endome-trioid andclear cell)

healthy con-trols: 20 endo-metriosis: 33

RT-qPCR miR-132 Suryawanshi etal. D54X X68D55X X

2013

miR-92 - Serum 50 EOC, no spe-cific subtype

50 RT-qPCR cel-miR-54 Guo et al. D56X X61D57X X 2013

miR-221 - Serum 96 serous: 70,other sub-types: 26

35 RT-qPCR mmu-miR-295 Hong et al. D58X X60 D59X X 2012

miR-200a, miR-200b, miR-200c

- Serum 28 HGSC 28 RT-qPCR miR-103 Kan et al.D60X X57D61X X 2012

Serum 28 15 RT-qPCR miR-142-3p Resnick et al.D62X X55D63X X 2009


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Table

I.(C

ontinued)

OverD2XXexpressed

miRNAs

UnderD3XXexpressed

miRNAs

Biological

specim

en

Ovarian

D4XXcancer

patie

nts

Ovarian

D5XXcancer

subtype

Health

ycontrols

Detectio

ntechnology

Reference

miRNA

Reference

Year

miR-21,miR-92,

miR-93,m

iR-

126,m

iR-29a

miR-155,miR-127,

miR-99b

EOC,nosp

e-

cificsubtype

miR-21,miR-

141,m

iR-

200a,miR-

200c,miR-

200b,miR-

203,m

iR-205,

miR-214

-Se

rum

exo

somes

50

Serous

healthycon-

trols:10

benigncase

s:10

Microarrays

conta

ining

probes

NR

Tayloretal.D64XX93D65XX

2008

EOC:epithelia

lovaria

ncancer,NR:notreported



miR-92, miR-93, miR-126, and miR-29a) and 3D329X XunderD330X Xex-

pressed (miR-155, miR-127, and miR-99b).D331X X55D332X X When

peripheral blood samples from ovarian cancer patients

that relapsed were analyzed using specific microarrays,

the elevated expression of miR-30c-1 and the decreased

expression of 3D333X X circulating cfmiRNAs (miR-342-3p,

miR-181a, and miR-450b-5p) D334X Xwere reported.D335X X56D336X XIn another

study, miRNAs from the miR-200 family, namely miR-

200a, miR-200b, and miR-200c were found significantly

overD337X Xexpressed in serum samples of serous ovarian cancer

patients when compared to healthy donors, using miR-

103 as a reference miRNA. miR-182 levels were also

assessed, however, no significant increment was reported

for patients in contrast with the ovarian cancer cell lines

examined.D338X X57D339X X These miR-200 family members were also

found elevated using U6 snRNA as a reference in a dif-

ferent study.D340X X58D341X XMoreover, increased levels of circulating

miR-200b were reported in pre- and post-treatment ovar-

ian cancer patients.D342X X59D343X XIn this study, Kapetanakis et al. eval-

uated levels of the tumor marker CA-125 and circulating

miR-200b in a group of 51 ovarian cancer patients, how-

ever, no significant correlation was found. Circulating

miR-200b was not associated with the FIGO stage,

whereas CA-125 levels significantly correlated with the

FIGO stage; however, CA-125 levels were very low in 3D344X X

out of 4D345X X early staged cases of the group and circulating

miR-200b levels were above averageD346X X59D347X XFurthermore, cir-

culating miR-221D348X X60D349X XmiR-92,D350X X61D351X Xand miR-21D352X X62,D353X X63D354X Xwere signif-

icantly upD355X Xregulated in serum samples of ovarian cancer

patients when compared to healthy women. Conversely,

miR-26a was found upD356X Xregulated in plasma samples of

ovarian cancer patients compared to healthy controls, in a

different study.D357X X64D358X XDownD359X Xregulation of miR-145 was con-

firmed in another study in ovarian cancer tissues, cell

lines, and serum samples.D360X X65D361X XThe diagnostic value of circu-

lating miR-145 levels in sera from patients with ovarian

cancer was determined with the corresponding levels in

healthy subjects, with evaluation using ROC curves (area

under curve, AUC = 0.82).D362X X66D363X X

Circulating miR-205 and let-7f were significantly

over D364X Xexpressed and under D365X Xexpressed, respectively, in a

large number of ovarian cancer patients using a Taq-

Man low density array; the diagnostic value of the

combination of these 2 D366X Xcirculating cfmiRNAs was esti-

mated using a ROC curve (AUC = 0.831). When the

levels of circulating miR-205 and let-7f were combined

with CA-125 values, a better diagnostic sensitivity was

observed. D367X X67 D368X X In endometriosis-associated (endometrioid

and clear cell) ovarian and serous ovarian cancer a dis-

tinct expression of specific miRNAs was detected in

each type of ovarian cancer when compared to healthy

women; more specifically, endometriosis-associated

ovarian cancer was characterized by the over D369X Xexpres-

sion of miR-16, miR-21, and miR-191, whereas serous


Table II. Prognostic and predictive value of circulating cell-free and exosomal miRNAs in ovarian cancerD66X X

Over-expressedmiRNAs

Under-expressedmiRNAs

Biologicalspecimen


Ovarian D68X Xcancersubtype

Detectiontechnology

Reference miRNA Prognosticsignificance

Reference Year

miR-200b, miR-1274a, miR-141, miR-200c

- Plasma 207 serous: 137, clearcell: 27, mixed:21, other sub-types: 22

RT-qPCR miR-220, miR-19b,U6 snRNA, miR-320

Patients receiv-ing Bevacizu-mab: PFS(miR-200c,P = 0.006)

Halvorsen et al. D69X X92D70X X 2017

miR-125b - Serum 135 HGSC: 89, clearcell: 24, endo-metrioid: 12,other subtypes:10

RT-qPCR miR-16 PFS (P D71X X= 0.035) Zhu et al. D72X X78D73X X 2017

- miR-148a Plasma 102 EOC, no specificsubtype

RT-qPCR U6 snRNA OS (PD74X X= 0.002) Gong et al.D75X X80D76X X 2016

- miR-199a Serum 70 mucinous: 36,serous: 17, othersubtypes: 17

RT-qPCR U6 snRNA OS (P = 0.030) Zuberi et al.D77X X76D78X X 2016

miR-373, miR-200a, miR-200b, miR-200c

- Serumexosomes

163 serous: 120, othersubtypes: 15,unknown: 28

RT-qPCR miR-484 OS (miR-200bPD79X X= 0.007, miR-200cP = 0.017)

Meng et al. D80X X94D81X X 2016

miR-7, miR-429 miR-25, miR-93 Serum 180 EOC, no specificsubtype

RT-qPCR miR-484 OS (miR-429,PD82X X= 0.011)

Meng et al. D83X X75D84X X 2015

miR-200b - Plasma 51 EOC, no specificsubtype

RT-qPCR miR-191 PFS (P D85X X= 0.003) Kapetanakis et al. D86X X59D87X X 2015

- miR-145 Serum 84 endometrioid: 24,serous: 18, clearcell: 17, mucin-ous: 12, mixed:13

RT-qPCR U6 snRNA OS (PD88X X= 0.039) Liang et al.D89X X66D90X X 2015

miR-200c, miR-141

- Serum 74 serous: 16, endo-metrioid: 15,clear cell: 14,mucinous: 12,undifferentiated:17

RT-qPCR NR OS (miR-200c, P D91X X< 0.001)

Gao et al. D92X X74D93X X 2015

miR-625-3p,miR-720, miR-1274a

19 miRs including:miR-19b, miR-223, miR-16, miR-150, miR-20a,miR-126, miR-1290

Plasma 42 Serous RT-qPCR Presurgical sam-ples: miR-320,miR-720, miR-1274b, U6 snRNAAll other sam-ples: 14 miRNAs

OS (miR-1290,PD94X X= 0.004)

Shapira et al. D95X X72D96X X 2014


ARTICLE

INPRESS

Transla

tionalR

ese

arch

Volume00

Giannopoulouetal

9


Table

II.(Continued)

Over-

expressed

miRNAs

Under-expressed

miRNAs

Biological

specim

en

Ovarian

D67XXcancer

patie

nts

OvarianD68XXcancer

subtype

Detectio

ntechnology

ReferencemiRNA

Prognostic

significance

Reference

Year

miR-205

let-7f

Plasm

a360

serous:179,e

ndo-

metrioid:86,

adenocarci-

noma:47,

mucinous:33,

clearcell:15

RT-qPCR

NR

PFS

(let-7f,

PD97XX=0.006)

Zhengetal.D98XX67D99XX

2013

miR-21

-Se

rum

94

serous:68,other

subtypes:26

RT-qPCR

U6snRNA

OS(PD100XX=0.006)

Xuetal.D101XX63D102XX

2013

miR-221

-Se

rum

96

serous:70,other

subtypes:26

RT-qPCR

mmu-m

iR-295

OS(PD103XX=0.005)

Hongetal.D104XX60D105XX

2012



ovarian cancer by the over D370X Xexpression of miR-16,

miR-191, and miR-4284. The diagnostic value of these

combinations was evaluated using ROC curves. D371X X68 D372X XCir-

culating cfmiR-99a was found down D373X Xregulated both in

serum and primary tissue ovarian cancer samples,

using RT-qPCR and U6 snRNA as a reference. D374X X69 D375X XAyaz

et al. examined the expression of a large number of

plasma circulating cfmiRNAs in ovarian cancer

patients and reported an increase and a decrease on

the expression levels of 8 D376X X (miR-191-5p, miR-206,

miR-548a-3p, miR-320a, miR-574-3p, miR-590-5p,

miR-34c-5p, and miR-106b-5p) and 4 D377X X (miR-150-5p,

miR-645, miR-30a-5p, and miR-19a-3p) miRNAs,

respectively, when compared to healthy controls. D378X X70D379X X

In another study, circulating let-7i-5p, miR-122,

miR-152-5p, and miR-25-3p were significantly under-

D380X Xexpressed in ovarian cancer patients compared to

patients with benign serous cystadenomas. D381X X71 D382X XShapira et

al. investigated the expression of a large number of

plasma circulating cfmiRNAs and reported a decrease

in the expression levels of 19 circulating cfmiRNAs in

ovarian cancer patients when compared to healthy con-

trols and an increment in the expression levels of 3 D383X X

miRNAs (miR-625-3p, miR-720, and miR-1274a) in

patients’ plasma samples.D384X X72 D385X X Three miRNAs (miR-22,

miR-93, and miR-451) were significantly over D386X Xex-

pressed and one (miR-106b) was under D387X Xexpressed in

serum samples of ovarian cancer patients. D388X X73 D389X XAccording

to another study, circulating miR-200c and miR-141

were over D390X Xexpressed in ovarian cancer patients. D391X X74 D392X X

When TaqMan PCR miRNA assays and miR-484 as

a reference miRNA were used for the estimation of the

expression levels of specific circulating cfmiRNAs in a

larger cohort of ovarian cancer patients, 2 D393X X miRNAs

(miR-7 and miR-429) were found over D394X Xexpressed and 2 D395X X

(miR-25 and miR-93) under D396X Xexpressed. It is important

to mention that both over D397X Xexpressed miRNAs, miR-7,

and miR-429, are involved in the EMT process. D398X X75 D399X X

Two circulating cfmiRNAs were investigated as

diagnostic biomarkers in ovarian cancer, using RT-

qPCR and U6 snRNA as a reference D400X X. D401X X76, D402X X77 D403X XThe levels of

miR-199a were significantly decreased in ovarian can-

cer patients when compared to healthy women. D404X X76 D405X X

Inversely, miR-125b was significantly over D406X Xexpressed

in ovarian cancer patients and its levels were signifi-

cantly correlated with FIGO stage, lymph node and dis-

tant metastasis. D407X X77 D408X X According to another study,

circulating cfmiR-125b was over D409X Xexpressed in serum

samples of ovarian cancer patients when compared to

benign controls. D410X X78 D411X X

Circulating cfmiRNAs were also investigated in a

large group of ovarian cancer patients, patients with

benign disease and healthy women. D412X X79 D413X XTaqMan miRNA

assays were used for the quantification of selected


Table III. Clinical significance of circulating cell-free and exosomal miRNAs in ovarian cancerD106X X

miRNAs Expression Clinical significance References

miR-21 OverD107X Xexpressed Diagnostic, Prognostic D108X X

55,D109X X

62,D110X X

63,D111X X

68,D112X X

93D113X X

miR-200a, miR-200b, miR-200c OverD114X Xexpressed Diagnostic, Prognostic D115X X

57-59,D116X X

74,D117X X

79,D118X X

83,D119X X

92-94D120X X


74,D123X X

92,D124X X

93D125X X

miR-125b OverD126X Xexpressed Diagnostic, Prognostic D127X X

77,D128X X

78D129X X


75D132X X

miR-221 Over-expressed Diagnostic, Prognostic D133X X

60D134X X

miR-26a OverD135X Xexpressed Diagnostic D136X X

64,D137X X

83D138X X

miR-205 OverD139X Xexpressed Diagnostic D140X X

67,D141X X

93D142X X


87D145X X


75D148X X


79,D151X X

94D152X X


87D155X X


55,D158X X

61D159X X


82D162X X

miR-1274a OverD163X Xexpressed Diagnostic D164X X

72,D165X X

92D166X X


55,D169X X

73D170X X

UnderD171X Xexpressed D172X X

75D173X X

let-7f UnderD174X Xexpressed Diagnostic, Prognostic D175X X

67D176X X

miR-145 UnderD177X Xexpressed Diagnostic, Prognostic D178X X

66D179X X

miR-199a UnderD180X Xexpressed Diagnostic, Prognostic D181X X

76D182X X

miR-25 UnderD183X Xexpressed Diagnostic, Predictive D184X X

71,D185X X

75D186X X

miR-148a UnderD187X Xexpressed Diagnostic, Prognostic D188X X

80D189X X

miR-1290 UnderD190X Xexpressed Prognostic D191X X

72D192X X


miRNAs and miR-484 was used as a reference. The

expression of 4 D414X XmiRNAs (miR-373, miR-200a, miR-

200b, and miR-200c) was significantly increased in

ovarian cancer patients when compared to healthy con-

trols; three of them (miR-200a, miR-200b, and miR-

200c) were also significantly increased in ovarian can-

cer patients compared to patients with benign disease.

Elevated levels of miR-373 were observed in advanced

stage patients exclusively, whilst these of miR-200a,

miR-200b, and miR-200c were observed in both, early

and advanced stage ovarian cancer. D415X X79 D416X XGong et al. inves-

tigated the expression levels of miR-148a in plasma

and observed a significant under D417X Xexpression in ovarian

cancer when compared to healthy controls. D418X X80 D419X XCirculat-

ing miR-376a was up D420X Xregulated in serum and primary

tumor ovarian cancer samples when compared to

healthy donors. D421X X81 D422X X

In a cohort of 168 HGSC patients, a significant

increase of the expression levels of 3 D423X XmiRNAs namely

miR-1246, miR-595, and miR-2278 in patients’ serum

samples when compared to healthy controls was

reported. According to ROC curves, miR-1246 was the

best diagnostic biomarker in HGSC ovarian cancer

(AUC = 0.89). D424X X82 D425X XYokoi et al. also recruited 155 ovarian

cancer patients and used a combination of 8 D426X Xcirculating

cfmiRNAs to establish a new diagnostic model for

early detection (Table I).D427X X83 D428X XIn 2 D429X Xvery recent studies, cir-

culating miR-1181 and miR-4314 were significantly

elevated in ovarian cancer samples when compared to

healthy donors D430X X84 D431X X and miR-193a-5p was significantly

decreased in serum samples.D432X X85 D433X X

Elias et al. developed a neural network of circulating

cfmiRNAs for ovarian cancer early detection by recruit-

ing 168 ovarian cancer patients of diverse histology and

healthy controls and identified a network of 14 differen-

tially expressed miRNAs. The proposed miRNA neural

network had 100% specificity and a significantly better

specificity and sensitivity compared to CA-125.D434X X86D435X X

When the diagnostic value of circulating cfmiRNAs

in combination with CA-125 in HGSC patients was

investigated using RT-qPCR, a significant over D436X Xexpres-

sion of 4 D437X XmiRNAs (miR-375, miR-210, miR-150-5p,

and miR-181-5p) in 2 D438X Xcohorts of HGSC patients com-

pared to healthy controls was reported; notably, a com-

bination of circulating miR-375 and CA-125 proved to

have a better diagnostic value, according to ROC

curves (AUC = 0.956). D439X X87 D440X X

A study on the role of circulating cfmiRNAs in clear

cell ovarian cancer and especially in disease recurrence

based on real-time PCR revealed that 4 D441X XmiRNAs were

found elevated in pre D442X Xoperative serum samples when

compared to post D443X Xoperative samples; interestingly,

miR-130a levels were increased in early recurrent dis-

ease, suggesting a possible role on the detection of can-

cer progression. D444X X88 D445X X Circulating cfmiR-135a-3p was

under D446X Xexpressed in serum samples of ovarian cancer

patients when compared to patients with benign disease

or healthy controls. D447X X89 D448X X




A very recent study, wherein the role of miR-590-3p

in the development epithelial ovarian cancer was inves-

tigated, revealed a possible involvement since over D449X Xex-

pression of miR-590-3p was observed in both, tissues

and plasma samples of ovarian cancer patients, com-

pared to patients with benign gynecological diseases. D450X X90D451X X

Prognosis. In a large number of studies, over- or

under D452X Xexpression of specific circulating cfmiRNAs was

significantly correlated with shorter OS and/or PFS,

indicating a possible prognostic value (Table II). More

specifically, over D453X Xexpression of circulating miR-21, D454X X63 D455X X

miR-221,D456X X60 D457X X miR-141 D458X X74 D459X X and miR-429, D460X X75 D461X X and under D462X Xex-

pression of miR-200c, D463X X74 D464X XmiR-1290 D465X X72 D466X XmiR-145, D467X X66 D468X XmiR-

199a D469X X76 D470X X and miR-148a, D471X X80 D472X Xwere correlated with shorter

OS. Additionally, increased levels of miR-200b D473X X59] and

miR-125b, D474X X78 D475X Xand decreased levels of let-7f D476X X67 D477X Xand miR-

135a-3p D478X X89 D479X X were significantly associated with shorter

PFS.

Response to treatment. The role of circulating cfmiR-

NAs in predicting treatment response was first evalu-

ated in a phase II clinical trial (NCT00477386, www.

clinicaltrials.gov). D480X X91 D481X X Patients with recurrent and plati-

num-resistant ovarian cancer were treated with the

hypomethylating agent decitabine, followed by carbo-

platin. The expression levels of 78 circulating cfmiR-

NAs were measured before and after the first cycle of

treatment; the subgroups of responders and non D482X Xres-

ponders were characterized by a different profile of

miRNAs, as shown in Table II. Interestingly, when all

patients’ data were studied together, 3 D483X X circulating

cfmiRNAs namely miR-616, miR-532-3p, and miR-

148b-5p were significantly over D484X Xexpressed in respond-

ers, while under D485X Xexpression of miR-148b-5p was signif-

icantly correlated with worse PFS. D486X X91 D487X X

In another recent study, the addition of Bevacizumab

to standard chemotherapy was investigated in a large

group of ovarian cancer patients (n = 207); the

increased level of circulating miR-200c was associated

with worse PFS in the subgroup of patients who addi-

tionally received Bevacizumab. D488X X92 D489X X

CIRCULATING EXOSOMAL miRNAs

The first study on circulating exosomal miRNAs in

ovarian cancer was performed ten years ago. D490X X93 D491X XCircu-

lating exosomes were extracted from serum of patients

with serous ovarian cancer, patients with benign dis-

ease and healthy women, using an anti-EpCAM assay.

A large number of exosomal miRNAs was examined,

but only 8 D492X X (miR-21, miR-141, miR-200a, miR-200c,

miR-200b, miR-203, miR-205, and miR-214) were

found elevated both, in serum samples and paired pri-

mary tumors. These miRNAs were not detected in

healthy controls and patients with benign disease had a

significantly different profile compared to ovarian can-

cer patients, indicating a possible clinical value of

these circulating exosomal miRNAs in the early diag-

nosis of ovarian cancer.D493X X93 D494X X

In another study, circulating exosomal miRNAs

were also investigated in a large group of ovarian can-

cer patients, patients with benign disease and healthy

women. D495X X94D496X XSpecific miRNA TaqMan assays were used

for the quantification of selected miRNAs and miR-

484 was used as reference miRNA. Four miRNAs

namely miR-373, miR-200a, miR-200b, and miR-200c

had significantly over D497X Xexpressed in ovarian cancer

patients compared to healthy controls, with D498X X3 D499X Xof them

(miR-200a, miR-200b, and miR-200c) being signifi-

cantly increased in ovarian cancer patients compared

to patients with benign disease. The levels of exosomes

in total were also increased in ovarian cancer patients

compared to the other 2 D500X Xgroups. All these findings sug-

gest a diagnostic value of the above circulating exoso-

mal miRNAs. D501X X94 D502X X

Ying et al. studied the expression of miR-222-3p in

primary tumors and serum samples of patients with

epithelial ovarian cancer, as well as in ovarian cancer

cell lines and reported a significant up D503X Xregulation of cir-

culating exosomal miR-222-3p in serum samples when

compared to healthy controls. They also report an over-

D504X Xexpression of miR-222-3p in exosomes derived from

ovarian cancer cells and macrophages; their subsequent

experiments suggested a possible role of miR-222-3p

in the polarization of tumor-associated macrophages. D505X X95 D506X X

In another study, a number of circulating miRNAs was

evaluated in exosomes isolated from serum and in

paired tissue samples of ovarian cancer patients, using

RT-qPCR and U6 snRNA as reference; only circulat-

ing exosomal miR-101 was significantly under D507X Xex-

pressed in serum exosomes compared to healthy

controls. D508X X96 D509X X

So far, only one study suggested a prognostic value

of circulating exosomal miRNAs in ovarian cancer; the

elevated levels of both, miR-200b and miR-200c, were

significantly associated with worse OS. Interestingly,

the increased levels of miR-200b and miR-200c also

significantly correlated with CA-125 levels. D510X X94 D511X X

CONCLUSIONS

Despite the increasing number of research and clini-

cal studies, ovarian cancer still has very low survival

rates among all gynecological malignancies. D512X X1 D513X X This

could be attributed to the lack of effective tumor bio-

markers; especially to the absence of an FDA-cleared

biomarker for the early detection of this disease. CA-

125 and HE4 are FDA-approved predictive biomarkers

that are also used for the detection of recurrent disease

ctgov:NCT00477386

http://www.clinicaltrials.gov

http://www.clinicaltrials.gov



after the completion of first-line treatment. D514X X97 D515X X In addi-

tion to the CA-125 and HE4 tumor biomarkers, a num-

ber of supplementary algorithms are also developed;

the risk of ovarian malignancy algorithm D516X X is FDA-

approved and combines the levels of CA-125 and HE4

with menopausal status to generate a score that indi-

cates the likelihood of malignancy in adnexal mass

patients. D517X X98 D518X X However, none of these were appropriate

for early diagnosis of the disease and, most impor-

tantly, for population screening.

The clinical significance of CTCs and ctDNA has

been investigated in many types of cancer, D519X X99 D520X X including

ovarian cancer. However, no standard methods are

used for the isolation and detection in the bloodstream

and only a few studies recruited large cohorts of ovar-

ian cancer patients.

Circulating cfmiRNAs and circulating exosomes can

serve as tumor biomarkers with the potential of provid-

ing information on early diagnosis, prognosis, response

to therapy and development of chemo D521X Xresistance. In

ovarian cancer, a large number of studies revealed the

diagnostic and prognostic potential of circulating

cfmiRNAs and exosomes; additional studies that will

be focused on their role in predicting response to treat-

ment and chemo D522X Xresistance development would be of

great interest.

Liquid biopsy approaches are minimally invasive

and allow for easily tolerated serial measurements dur-

ing treatment. However, there is a lot to be done in

terms of pre D523X Xanalytical and post D524X Xanalytical conditions,

quality control, and validation of the assays used for

circulating cfmiRNAs and exosomes isolation, detec-

tion, and characterization, before their implementation

in clinical practice. In conclusion, circulating cfmiR-

NAs and exosomes constitute a main component of liq-

uid biopsy analysis, with the potential of contributing

to the development and establishment of personalized

medicine protocols in ovarian cancer patients.

ACKOWLEDGMENTS

All authors have read the journal’s policy on disclo-

sure of potential conflicts of interest and have declared

no conflicts of interest. All authors have read the jour-

nal’s authorship agreement and declare that the manu-

script has been reviewed and approved by all named

authors.

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