Prognostic Significance of Soluble Fas and Soluble Fas Ligand in Serum of Patients with Complete Hydatidiform Moles

Prognostic Significance of Soluble Fas and Soluble Fas Ligand inSerum of Patients with Complete Hydatidiform MolesSimmi Soni1, Gayatri Rath1, Chandra P. Prasad1, Sudha Salhan2, Ashwini Kumar Mishra3, Sunita Saxena3

1Department of Anatomy, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India;2Department of Obstetrics and Gynaecology, Vardhman Mahavir Medical College & Safdarjung Hospital, New Delhi, India;3Institute of Pathology – ICMR, New Delhi, India

Introduction

Hydatidiform mole (HM), a relatively rare pregnancy-

associated disorder, is still an enigma regarding its

physiopathology and progression. Based on histopath-

ological and clinical criterion, it is classified into two

entities: partial hydatidiform mole (PHMs) and com-

plete hydatidiform mole (CHMs).1 HM possesses the

potential to evolve to persistent trophoblastic disease

(PTD) requiring therapeutic interventions and the risk

is considerably higher for CHMs.2 A combination of

abnormally functioning genes is involved in the path-

ogenesis of molar pregnancy, either contributing to or

as a consequence of the deranged apoptosis. The

Keywords

Apoptosis, complete hydatidiform moles,

placenta, sFas, sFas L

Correspondence

Gayatri Rath, Department of Anatomy,

Vardhman Mahavir Medical College &

Safdarjung Hospital, New Delhi 110029, India.

E-mail: [email protected]

Submitted October 10, 2010;

accepted January 15, 2011.

Citation

Soni S, Rath G, Prasad CP, Salhan S, Mishra

AKumar, Saxena S. Prognostic significance of

soluble Fas and soluble Fas ligand in serum of

patients with complete hydatidiform moles.

Am J Reprod Immunol 2011

doi:10.1111/j.1600-0897.2011.00988.x

Problem

Despite of advances in diagnosis and staging, the prognosis of hydatidi-

form mole (HM) remains intricate. HM possesses the substantial risk of

developing persistent trophoblastic disease (PTD), which is considerably

high for complete hydatidiform moles (CHMs). Significance of serum

soluble Fas (sFas) and soluble FasL (sFasL) has been observed in various

malignancies; however, there is no report till date on HM.

Method of study

The serum levels of sFas and sFasL were measured using enzyme-linked

immunosorbent assay in 62 patients with CHMs and 64 healthy con-

trols. The protein concentrations were also correlated with clinicopatho-

logical parameters, b-hCG level, and clinical outcome.

Results

The serum sFas and sFasL levels in patients with CHM were significantly

higher than those in control group (mean ± SD: 703.497 ± 491.759 ver-

sus 348.141 ± 175.24; P < 0.004 and 31.17 ± 18.758 versus 18.802 ±

6.775; P < 0.0001, respectively). Patients who progressed to PTD demon-

strated higher sFas and sFasL concentrations than those who regressed

spontaneously (794.211 ± 415.892 versus 446.69 ± 161.382; P < 0.046

and 37.55 ± 20.337 versus 22.763 ± 6.52; P < 0.011, respectively). Fur-

thermore, significant associations were observed among sFas, sFasL, and

b-hCG levels (P < 0.0001 for all associations).

Conclusion

Production of sFas and sFasL may play a crucial role in progression of

CHM and may serve both as prognostic tool and therapeutic target in

improving the clinical outcome.

A J I 9 8 8 B Dispatch: 28.1.11 Journal: AJI CE: Vinoth

Journal Name Manuscript No. Author Received: No. of pages: 7 PE: Sangeetha.C

SHORT COMMUNICATION

American Journal of Reproductive Immunology (2011)

ª 2011 John Wiley & Sons A/S 1

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molecular pathway leading to apoptotic cell death is

initiated by several proteins that act as death factors.

The Fas ⁄Fas ligand system represents one of the best-

characterized death factor systems.3

Both Fas and Fas ligand are membrane proteins,

belonging to tumor necrosis factor family, and exist

in two isoforms, the transmembrane and the soluble

forms.3 The membrane isoform of Fas (mFas) is a

45-kDa protein containing a single transmembrane

region and induces apoptosis in normal or tumor

cells when bound by FasL.4 The soluble isoform

(sFas) lacks the transmembrane domain and is usu-

ally generated by differential splicing of the tran-

script or transformed from mFas.5 It is thought to

block Fas-mediated apoptosis by binding and subse-

quently inactivating FasL, acting as a physiological

antagonist for Fas.6 Elevated levels of sFas have been

reported in the serum of patients with bladder,

breast, renal cell, hepatocellular, prostate, and gyne-

cological cancers.7–12

The membrane-bound FasL (mFasL) is a 37-kDa

protein, expressed by activated T lymphocytes, natu-

ral killers cells, and a few immunoprivileged tis-

sues.13 The mFasL may undergo proteolytic cleavage

by a specific matrix metalloproteinase like enzyme

to liberate the soluble form (sFasL). Human sFasL is

a 26-kDa glycoprotein consisting of an extracellular

region for binding to Fas.14 Both membranous

(mFasL) as well as soluble forms of FasL (sFasL) bind

to Fas and transduce an apoptotic signal in Fas-

expressing cells. There are controversies regarding

the precise physiological role of sFasL. Although

human sFasL is shown to stimulate apoptosis, this

activity is generally regarded to be lesser than that of

membrane-associated FasL.13 High serum concentra-

tions of sFasL have been observed in patients with

natural killer cell lymphoma, non-hematopoietic

malignancy, hepatocellular carcinoma, and graft-ver-

sus-host disease.15

Although there is limited data in the literature

regarding the expression of membranous Fas and

membranous FasL in pathologic placentae including

HM,16–19 to the best of our knowledge, there is no

report to date that demonstrates the significance of

their soluble forms (sFas and sFasL) in patients with

CHMs. Hence, in the present study, we investigated

the serum concentration of sFas and sFasL in

women with CHMs and to assess the biological and

clinical relevance of these circulating proteins in

HMs.

Materials and methods

Study Population

A total of 128 (62 + 64) cases were enrolled in the

study. The case group included 62 untreated patients

with CHMs (ranging in age from 24 to 35 years, with

a median age of 24 years) who were admitted to

Department of Obstetrics & Gynaecology (VMMC &

Safdarjung Hospital, New Delhi, India) between 2006

and 2009. They presented with ultrasonographic

abnormalities indicative of molar pregnancy, and the

diagnosis was confirmed with histological examina-

tion of samples after suction evacuation. All the

patients included in the study were CHMs, because

of inadequate number of PHMs available. The patient

data included gestational age, gravidity, parity, abor-

tions, and obstetrics history (Table I). The gestational

age based on the last menstrual period, ranged from

8 to 20 weeks (median age 14 weeks). The control

group consisted of 64 gestational matched healthy

pregnant women (8–20 weeks) without any renal,

heart, or vascular diseases and who had no previous

history of infectious, allergic, autoimmune, or other

systemic diseases. None of the patients from control

group had any smoking ⁄ alcohol ⁄ tobacco habits.

The study was approved by the institutional ethics

committee and each subject enrolled in the study

gave informed written consent. Follow-up b-hCG

assays were carried out in patients with CHMs at

weekly intervals for 3 months and then monthly for

1 year. Follow-up was accessible in 30 of 62 patients,

and among 30 cases, 12 underwent spontaneous

regression whereas 18 progressed to PTD.

Enzyme-Linked Immunosorbent Assay (ELISA)

Peripheral venous blood was drawn by venipuncture

from each patient before suction evacuation and the

controls. Serum was obtained by centrifugation

within 45 min of blood collection and stored at

)80�C until assayed. sFas ⁄ sFasL concentrations were

measured using a sandwich ELISA and b-hCG levels

were evaluated by Direct ELISA with commercially

available ELISA kits (Human sFas Immunoassay

DFS00; R&D systems 1; human sFas Ligand Immuno-

assay DFL00; R&D Systems; b-hCG ELISA kit; Diam-

etra diagnostic kits 2), as per the manufacturer’s

instructions. The minimal detectable limits for sFas

and sFasL were 20.0 and 2.66 pg ⁄mL, respectively.

SONI ET AL.


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Briefly, the 96-well microplate coated with mouse

monoclonal antibody against sFas ⁄ sFasL ⁄b-hCG was

blocked with assay diluent provided. Then, the stan-

dards and diluted ⁄neat serum samples were added in

duplicate to the wells and incubated at room tem-

perature for 2 hr. After each well was aspirated and

washed five times with wash buffer, conjugate

(horseradish peroxidase–conjugated secondary anti-

body) was added to each well and the plate was

incubated for 2 hr. Later, each well was again aspi-

rated ⁄washed in a similar manner and incubated

with substrate solution (tetramethylbenzene and

hydrogen peroxide) in dark for 30 min. Finally, the

reaction was stopped by adding stop solution (1 N

sulfuric acid). The optical density was measured

using a spectrophotometric microtiter plate reader

(Powerwave XS, MQX200R; Bioteck Instruments

Inc, USA3 ) at 450 nm. The concentration of protein

was determined from a calibration curve built using

reference standards.

Statistical Analysis

The data of sFas and sFasL levels were expressed as

mean ± standard deviation (mean ± SD) and ana-

lyzed using spss (18.0) statistics software (SPSS Inc.,

Chicago, IL, USA). Disparity in sFas ⁄ sFasL levels

between patients with CHMs versus healthy controls

and correlation between clinicopathological parame-

ters versus protein concentration was determined

with Wilcoxon W-test and Mann–Whitney U-test

[Asymp. Sig. (two-tailed)]. The associations among

proteins (sFas, sFasL and b-hCG) were explored

using Pearson correlation test (two-tailed). The

P-values <0.05 were regarded as significant for

Mann–Whitney U-test [Asymp. Sig. (two-tailed)]

and <0.01 was considered significant for Pearson

correlation test (two-tailed).

Results

Correlations between the clinicopathological parame-

ters and sFas ⁄ sFasL levels are summarized in Table I.

There was no statistically significant association

between serum concentration of proteins (sFas and

sFasL) with maternal age, gestational age, gravidity,

parity, bad obstetrics history, or habits (smok-

ing ⁄ alcohol ⁄ tobacco). However, both sFas and sFasL

values were significantly higher in patients with the

incidence of prior abortion (mean ± SD: 618.412 ±

418.233 versus 1359.708 ± 381.994; P < 0.001 and

27.243 ± 12.630 versus 61.629 ± 27.124; P < 0.003,

respectively).

The concentrations of sFas and sFasL in serum of

women with CHMs and healthy controls are shown

in Table II. The results showed increased serum sFas

and sFasL levels in patients with CHMs compared

to the controls (mean ± SD: 703.497 ± 491.759

pg ⁄mL versus 348.141 ± 175.24 and 31.17 ± 18.758

pg ⁄mL versus 18.802 ± 6.775, respectively), and this

increase in the serum levels of sFas and sFasL was

found statistically significant (P < 0.004 and P <

Table I Correlation of clinicopathological parameters with soluble FasL and soluble Fas (sFas) levels in hydatidiform mole (HM)

Clinicopathological

parameters No. of Cases

sFas L (pg ⁄mL)

P-value

sFas (pg ⁄mL)

P-valueMean ± SD Mean ± SD

Maternal age <30 52 29.862 ± 16.709 0.116 674.763 ± 508.524 0.242

‡30 10 37.972 ± 27.227 868.717 ± 363.246

Gestational age (wks) 8–12 12 29.936 ± 23.457 0.246 518.698 ± 472.630 0.266

13–20 50 31.200 ± 17.750 760.836 ± 497.122

Parity Primi 23 32.922 ± 19.747 0.945 622.856 ± 472.471 0.287

Multi 39 31.730 ± 19.513 813.425 ± 501.834

Abortions Nil 51 27.243 ± 12.630 0.003 5* 618.412 ± 418.233 0.001*

‡1 11 61.629 ± 27.124 1359.708 ± 381.994

Bad obstetrics history No 53 27.646 ± 13.478 0.08 647.064 ± 442.139 0.12

Yes 9 33.871 ± 25.190 729.368 ± 344.993

Habits (tobacco ⁄ alcohol) No 50 33.172 ± 20.262 0.087 729.871 ± 495.565 0.318

Yes 12 22.829 ± 5.307 611.189 ± 487.703

Mann–Whitney U-test & Wilcoxon W-test [Asymp. Sig. (two-tailed)]; *significant. Bad obstetric history includes history of tubal pregnancy ⁄ pre-

natal death ⁄ still birth ⁄ ovarian cyst ⁄multiple fibroids ⁄ previous HM.

sFAS AND sFASL IN COMPLETE HYDATIDIFORM MOLES



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0.0001). In addition, we found significant association

between both sFas and sFasL concentrations and dis-

ease progression (P < 0.046 and P < 0.011, respec-

tively) (Table III). Patients who progressed to PTD

had higher sFas and sFasL concentration than

those who regressed within 3 months (mean ± SD:

794.211 ± 415.892 pg ⁄mL versus 446.69 ± 161.382

and 37.55 ± 20.337 pg ⁄mL versus 22.763 ± 6.52,

respectively). Furthermore, the statistical analysis of

the evaluated results also revealed a significant posi-

tive correlation between levels of sFas, sFasL, and b-

hCG in the serum of study group (P < 0.0001 for all

associations) (Table IV).

Discussion

The Fas ⁄FasL system is essential for key physiological

functions in a variety of organs, including the main-

tenance of immune homeostasis.20 The expression

and signaling by Fas and its ligand (FasL, CD95L)

are tightly regulated through a variety of mecha-

nisms, one of which is postulated to be the produc-

tion of their soluble forms (sFas and sFasL).21 It has

been observed that circulating sFas and sFasL are

abnormally activated in variety of tumors.

Few studies have demonstrated the expression of

membranous Fas (mFas) and mFasL in placental

tissue of patients with HMs;22–24 however, to the best

of our knowledge, there is no literature on their solu-

ble forms. Owing to an important role of the sFas and

sFasL in tumor progression, the aim of the present

study was to estimate the levels of serum sFas and

sFasL in women with CHMs. Our results revealed

remarkable increase in the serum levels of both

sFas and sFasL in patients with CHMs in comparison

with the control group (Table II). Further, the

Table II Soluble Fas (sFas) and soluble FasL concentrations in hydatidiform mole (HM) and controls

Soluble markers

Control (n = 64) HM (n = 62)


sFas Ligand 18.802 ± 6.775 (pg ⁄mL) 31.170 ± 18.758 (pg ⁄mL) 0.0001*

sFas 348.141 ± 175.240 (pg ⁄mL) 703.497 ± 491.759 (pg ⁄mL) 0.004*

Mann–Whitney U-test and Wilcoxon W-test [Asymp. Sig. (two-tailed)]; *significant.

Table III Soluble Fas (sFas) and soluble FasL concentrations in progressed and regressed cases of hydatidiform mole

Progressed (n = 18) Regressed (n = 12)


sFas Ligand 37.550 ± 20.337 (pg ⁄mL) 22.763 ± 6.520 (pg ⁄mL) 0.011*

sFas 794.211 ± 415.892 (pg ⁄mL) 446.690 ± 161.382 (pg ⁄mL) 0.046*

b-hCG 120802.0 ± 114267.3 (pg ⁄mL) 7146.939 ± 8636.523 (pg ⁄mL) 0.0001*

Mann–Whitney U-test & Wilcoxon W-test [Asymp. Sig. (two-tailed)]; *significant.

Table IV Positive correlations among different soluble markers

No. of cases sFas ligand sFas b-hCG

sFas Ligand 62 1 0.753* (0.0001) 0.701* (0.0001)

sFas 62 0.753* (0.0001) 1 0.584* (0.0001)

b-hCG 62 0.701* (0.0001) 0.584* (0.0001) 1

sFas, soluble Fas. Pearson correlation test (two-tailed); *Correlation is highly significant at the 0.01 level.

SONI ET AL.



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concentrations of these soluble proteins also corre-

lated with the clinical outcome of disease (Table III).

High serum sFas and sFasL concentrations have been

observed in patients with hepatocellular, renal cell,

breast, endometrial, uterine, and ovarian carcinoma,3,25

which is in accordance with the results of our study.

The origin of sFas in the serum of patients remains

uncertain, although there are three proposed theo-

ries; sFas may be produced by the tumor itself or by

peripheral blood lymphocytes. The third theory

states that the surrounding stromal cells may pro-

duce sFas in response to the tumor or immune acti-

vation.25 As documented by several studies that sFas

increases in the serum of patients in a manner

directly related to tumor stage and burden,6 the first

theory appears to be more pertinent. Production of

sFas in tumor patients may be a key mechanism to

inhibit Fas-mediated apoptosis. As sFas has been

shown to bind FasL and competitively antagonize

Fas signaling, it may provide a key protective signal

that helps tumor cells avoid apoptosis in their hostile

microenvironment.6 In the present study, the ele-

vated levels of sFas observed in CHMs may prevent

and protect Fas-bearing trophoblasts cells from

undergoing apoptosis by FasL expressed on maternal

immune cells. Consequently, the activated immune

cells remain at the interface leading to immune tol-

erance and tumor survival (Fig. 1). Our previous

report on mFas and mFasL in placental tissue of

CHMs supports the current findings.26

The sFasL was originally thought to induce apop-

tosis in a manner similar to membrane-associated

FasL (mFasL). Yet, there have been many succeeding

reports supporting the disparity between sFasL and

mFasL regarding apoptosis induction.13 It is thought

that sFasL is less potent at inducing apoptosis than

membrane-bound FasL; however, the induction

effect varied with the cell types.15,25 The significantly

elevated level of sFasL and its positive correlation

with disease progression in CHMs denotes that the

sFasL is competent enough at inducing apoptosis in

maternal immune cells of patients with HM.

Previous reports have demonstrated that the mem-

brane-bound Fas ligand (mFasL) in CHMs serves as a

potential mechanism to inhibit maternal immune

function by inducing apoptosis of activated lympho-

cytes.22,23 In the present study, we observed signifi-

cant increase in the concentration of sFasL in serum

samples of CHM, which correlated with disease pro-

gression. Here, it has been presumed that this solu-

ble mediator (sFasL) may spread the apoptosis

induction effect by inducing apoptosis of Fas-

expressing lymphocytes even without direct cell–cell

contact. The study suggests that the CHMs that

release sFasL apart from expressing mFasL may have

an additional advantage to evade maternal immune

surveillance by inducing apoptosis of distant lym-

phocytes as well (Fig. 1). Hence, our data support

the hypothesis of others who have proposed a ‘coun-

terattack model’ suggesting that tumor cells use FasL

Fig. 1 Schematic diagram illustrating the role

of sFas and soluble FasL (sFasL) in pathogene-

sis and progression of hydatidiform moles.

The FasL is cleaved to sFasL and prevented

from binding to Fas receptor on molar tropho-

blast. The Fas receptor in turn gets converted

to sFas, which inhibits both FasL and sFasL

from binding to Fas expressed on molar tro-

phoblast by competing for binding. In con-

trast, both FasL and sFasL in the

microenvironment bind to Fas-expressing

immune cells causing them to undergo apop-

tosis, thus promoting tumor cell survival.6

LOW

RESOLUTIO

NCOLOR

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and sFasL as cytolytic effectors to kill Fas-expressing

activated lymphocytes, indicating that both may

offer subsequent tumor survival advantage.27–29

It is well established that the trophoblasts from

HM overexpress and secrete higher amounts of hCG

compared with normal trophoblasts and hCG is sug-

gested to have a role in trophoblast transformation,

growth, and invasion in gestational trophoblastic

neoplasias.30 Studies have shown that hCG stimu-

lates FasL expression in human endometrium and

that the hCG-treated endometrial cells induce an

increase in T-cell apoptosis.31 In the current study,

both sFas and sFasL levels revealed a positive associ-

ation with serum b-hCG concentration (P < 0.0001

and P < 0.0001, respectively) (Table IV), thus indi-

cating the regulatory effect of hCG on these soluble

markers in CHMs. We postulate that hCG may be a

link in evasion of peritrophoblastic immune surveil-

lance, by regulating sFasL and facilitate tumor growth

by regulating sFas, as hCG is known to influence

both maternal immune tolerance and Fas–Fas ligand

system.31 Furthermore, the current study demon-

strated a significant positive correlation between sFas

and sFasL (P < 0.0001), suggesting that both the pro-

teins are functioning in a harmonized manner, plau-

sibly toward tumor cell progression (Table IV).

In conclusion, sFas and sFasL play a significant

role in pathogenesis and progression of CHMs and

serves as a fertile ground for future research. As the

pathophysiological roles for sFas and sFasL emerge

in hydatidiform mole, these proteins may become

new targets in both detection and intervention. The

sFas and sFasL levels may prove to be useful in pre-

dicting those cases which are likely to progress, as

well as in the selection of therapeutic strategies, to

improve the clinical outcome of patients with CHMs.

However, further larger studies are warranted to

authenticate the current findings.

Acknowledgments

The study was supported by Indian Council of Medi-

cal Research (ICMR), New Delhi, India (Grant no:

5 ⁄13 ⁄16 ⁄2007-NCD-III). We acknowledge our tech-

nical staff; Miss Divya, Mr Dinesh, and Mr Musheer

for their assistance.

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Author Query Form

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