Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during...

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Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and

falls during treatment

Running title: Kisspeptin is raised in trophoblastic neoplasia

Waljit S. Dhillo1, Philip Savage2, Kevin G. Murphy1, Owais B. Chaudhri1, Michael

Patterson1, Gurjinder M. Nijher1, Vanessa M. Foggo2, Garin S. Dancey2, Hugh Mitchell2,

Michael J. Seckl2, Mohammad A. Ghatei1 & Stephen R. Bloom1

1Department of Metabolic Medicine, Hammersmith Hospital, Imperial College London,

UK & 2Department of Medical Oncology, Charing Cross Hospital, Imperial College

London, UK.

Corresponding author: Prof SR Bloom, Department of Metabolic Medicine,

Hammersmith Hospital, Imperial College London, 6th Floor Commonwealth Building,

Du Cane Road, London, W12 ONN, UK. Tel: +44 208 383 3242. Fax: +44 208 383

3142. E-mail: [email protected].

of 36Articles in PresS. Am J Physiol Endocrinol Metab (June 6, 2006). doi:10.1152/ajpendo.00555.2005

Copyright © 2006 by the American Physiological Society.

mailto:[email protected]

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Abstract Kisspeptin is a 54 amino acid peptide, encoded by the anti-metastasis gene

KiSS-1, that activates the G-protein-coupled receptor, GPR54. The kisspeptin/GPR54

system is critical to normal reproductive development. KiSS-1 gene expression is

increased in the human placenta in normal and molar pregnancies. Circulating kisspeptin

is dramatically increased in normal pregnancy but levels in gestational trophoblastic

neoplasia (GTN) have not previously been reported. The present study was designed to

determine if plasma kisspeptin levels are altered in patients with malignant GTN. Thirty

nine blood samples were taken from 11 patients with malignant GTN at presentation,

during and following chemotherapy. Blood was also sampled from non-pregnant and

pregnant volunteers. Plasma kisspeptin immunoreactivity (-IR) and human chorionic

gonadotrophin (hCG) concentrations were measured. Plasma kisspeptin-IR concentration

in non-pregnant (n=16) females <2 pmol/L. Plasma kisspeptin-IR in females in the first

trimester of pregnancy (n=13) was 803 ± 125 pmol/L, in the third trimester of pregnancy

(n=7) was 2483 ± 302 pmol/L and day 15 post partum (n=7) was <2 pmol/L. Plasma

kisspeptin-IR and hCG concentrations in patients with malignant GTN were elevated at

presentation and fell during and following treatment with chemotherapy in each patient

(mean plasma kisspeptin-IR: pre-chemotherapy 1363 ± 1076 pmol/L vs. post-

chemotherapy <2 pmol/L, p<0.0001. Mean plasma hCG: pre-chemotherapy 227191 ±

152354 U/L vs. post-chemotherapy 2 U/L, p<0.0001). Plasma kisspeptin-IR strongly

positively correlated with plasma hCG levels (r2=0.99, p<0.0001). Our results suggest

that measurement of plasma kisspeptin-IR may be a novel tumor marker in patients with

malignant GTN.

Keywords: kisspeptin, metastin, hCG, GPR54, gestational trophoblastic neoplasia.

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Introduction

Kisspeptin is a 54 amino acid peptide, encoded by the KiSS-1 gene (20, 21, 32) which

activates the previously orphan G-protein-coupled receptor, GPR54 (20). The

kisspeptin/GPR54 system has recently been shown to be critical to normal reproductive

development in rodents and humans. GPR54 deficient (GPR54-/-) mice have abnormal

sexual development, low circulating gonadotrophin concentrations and are infertile (12,

39). GPR54 mutations have been shown to cause hypogonadotrophic hypogonadism in

humans (7, 39, 40). Kisspeptin potently stimulates the hypothalamo-pituitary-gonadal

(HPG) axis in rodents, primates and human males (9, 13, 25, 28, 29, 30, 41, 46). The

stimulatory effects of kisspeptin on the HPG axis appear to be mediated via the

hypothalamic gonadotrophin releasing hormone (GnRH) system (13, 17, 25, 26).

Hypothalamic KiSS-1 expression is regulated by circulating sex steroids in rodents and

primates, suggesting that kisspeptin is involved in the HPG negative feedback cycle (17,

28, 41, 43, 44).

KiSS-1 and GPR54 genes are highly expressed in the placenta (1, 21, 32) and endogenous

forms of kisspeptin 54, 14 and 13 amino acids in length have been isolated from human

placenta. The common C terminal decapeptide shared by these forms, kisspeptin-10, is

the minimum sequence necessary for receptor activation (20, 27, 32) and is secreted from

cultured human trophoblasts (1). All these kisspeptin fragments, including kisspeptin-10,

have a similar affinity and efficacy in vitro at the GPR54 (20). KiSS-1 and GPR54 gene

expression are highly expressed in the placenta of normal pregnant humans (1, 18, 35).

Circulating kisspeptin is low in healthy men and non-pregnant women, but dramatically

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increased in normal pregnancy, reaching a concentration approximately 7000 fold higher

in the third trimester compared to non-pregnant controls (14).

The kisspeptin/GPR54 system is also important in tumor biology. KiSS-1 was first

discovered as an anti-metastasis gene (21, 32). KiSS-1 suppresses metastasis in human

breast carcinomas (22), and KiSS-1 expression inversely correlates with increased

metastasis and/or cancer progression in gastric, esophageal and pancreatic cancer,

phaeochromocytoma, bladder cancer, melanoma and breast cancer (8, 15, 24, 31, 37, 42,

45). KiSS-1 therefore represents a potential marker to distinguish metastatic from non-

metastatic forms of specific cancers.

Placental KiSS-1 gene expression has also recently been shown to inversely correlate

with cancer progression in gestational trophoblastic disease (GTD) or neoplasia (GTN)

(18, 35). GTN comprises a number of disorders characterized by an abnormal

proliferation of placental tissue. Complete (CHM) and partial hydatidiform moles (PHM)

are the commonest form of the disease, occurring in 1-3 of every 1000 pregnancies. They

are best regarded as premalignant, as 16% of CHM and 0.5% of PHM transform with

time into the malignant conditions of invasive mole, choriocarcinoma (CC) or placental

site trophoblastic tumor (PSTT) (33, 38). The latter three diseases are often referred to as

malignant GTN or as gestational trophoblastic tumors.

All forms of GTN secrete human chorionic gonadotrophin (hCG) and serum

measurement of this hormone is extremely helpful in the diagnosis, staging and

subsequent assessment of the therapeutic response of malignant GTN. Following uterine

evacuation of a molar pregnancy, the serum hCG in most women returns to normal.

However, in those developing malignant change, the hCG plateaus and/or starts rising

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and requires urgent chemotherapy which is curative in virtually 100% of cases. The

onset of malignant change following a molar pregnancy can take some months to occur

and women on hCG surveillance have to avoid becoming pregnant. Clearly, earlier

detection of this malignant change would be helpful, as it would enable rapid selection of

patients for additional therapy. Consequently, the development of new markers for GTN

may provide better predictive models of trophoblastic behavior. Moreover, many existing

commercial assays for hCG detection are troubled by false positives (3), usually induced

by heterophile antibodies. Heterophile antibodies are IgG immunoglobulins raised against

poorly defined antigens from other species and can be found in 3–15% of healthy people.

Furthermore, these assays usually do not detect all the forms of circulating hCG produced

in GTN (5, 19). New markers might also help to reduce these problems.

We hypothesized that plasma kisspeptin-IR may be altered in patients with malignant

GTN since placental KiSS-1 gene expression is increased in normal and molar

pregnancies (18, 35) and circulating kisspeptin is dramatically increased in normal

pregnancy (14).

In the present study we used a specific radioimmunoassay (RIA) for human kisspeptin

and investigated kisspeptin-IR in human plasma in pregnant and non-pregnant volunteers

and in patients with malignant GTN at presentation, during and following chemotherapy.

Kisspeptin-IR in plasma was characterized using reverse phase fast protein liquid

chromatography (FPLC). We found that kisspeptin-IR was elevated in patients with

malignant GTN compared to controls and positively correlated with plasma hCG levels.

Chemotherapy treatment reduced kisspeptin-IR and hCG levels in each patient.

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Methods

Subjects

Eleven healthy females who had never been pregnant with normal menstrual cycles

(mean age 26.1 ± 2.2 years), five healthy females who had previously been pregnant with

normal menstrual cycles (mean age 29.1 ± 3.1 years), thirteen healthy women in the first

trimester (10.4 ± 0.5 weeks) of a normal pregnancy (mean age 30.7 ± 1.7 years), seven

healthy women at 38 weeks of a normal pregnancy and the same women 15 days post

partum (mean age 34.4 ± 1.1 years) and eleven female subjects diagnosed with invasive

mole (mean age 27.2 ± 3.4 years) participated in the study. Patients were diagnosed with

molar pregnancy if they had amenorrhea with a raised plasma hCG and an ultrasound

confirming a molar pregnancy. Patients were diagnosed with an invasive mole if they had

a persistently elevated or rising serum and urine hCG following evacuation of their molar

pregnancy. Blood was collected at first presentation (between 1 to 6 months after removal

of their molar pregnancy) and then during and following chemotherapy as their serum

hCG levels fell. Patients with malignant GTN had their disease risk assessed according to

the FIGO scoring system for GTN (16). Women scoring six or less (low risk) received

intramuscular methotrexate on alternate days for eight days, followed by six rest days,

with each course consisting of four injections (36). Women scoring seven or more (high

risk) received combination therapy with intravenous etoposide, methotrexate, dactomycin

for two days followed by vincristine and cyclophosphamide (EMA-CO) one week later.

The course was then repeated after six days. Treatment was continued, in all cases, until

the hCG level had returned to normal and then for a further six consecutive weeks (36).

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Subjects gave written informed consent and ethical approval was obtained from the

Hammersmith and Queen Charlotte’s & Chelsea Hospitals Research Ethics Committee

(number 04/Q0406/80). Studies were performed in accordance with the Declaration of

Helsinki. Blood was taken from all subjects between 9am and noon.

(i) Determination of plasma kisspeptin-IR in healthy female volunteers

Eleven healthy females with regular menstrual cycles, not taking any oral contraception

had blood sampled during the follicular and luteal phases of their menstrual cycle. Five

healthy females who had previously been pregnant (mean 2.9 ± 2.4 years post partum)

with normal menstrual cycles also had blood sampled in the follicular phase of their

menstrual cycle. Thirteen healthy pregnant women had blood sampled during the first

trimester (10.4 ± 0.5 weeks) of their pregnancy. Blood was also sampled from seven

healthy pregnant females at both 38 ± 0.8 weeks gestation of normal pregnancy and day

15 ± 6 post partum. Ten ml of blood was taken and collected into lithium-heparin tubes

(LIP Ltd., Cambridge, UK) containing 5000 kallikrein inhibitor units (0.2 ml) aprotinin

(Trasylol, Bayer, Newbury, UK) and stored on ice. After centrifugation, plasma was

immediately separated and stored at -20oC until measurement of kisspeptin-IR in all

subjects and hCG in the pregnant subjects.

(ii) Determination of plasma hormones in patients at diagnosis of malignant GTN,

during treatment and post chemotherapy

Blood was sampled from patients with malignant GTN at first presentation, during

chemotherapy and following chemotherapy once the patient’s plasma hCG had fallen

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back into the normal range for non-pregnant females. The average length of time between

the collection of the second blood sample and the initiation of chemotherapy was 21.1 ±

2.8 days. The average time post chemotherapy at which a blood sample was taken was

14.3 ± 1.4 days. Blood was collected into lithium-heparin tubes (LIP Ltd., Cambridge,

UK) containing 5000 kallikrein inhibitor units (0.2 ml) aprotinin (Trasylol, Bayer,

Newbury, UK) and stored on ice. After centrifugation, plasma was immediately separated

and stored at -20oC until measurement of kisspeptin-IR, hCG, progesterone and

oestradiol.

(iii) Characterization of kisspeptin-IR in human plasma

Kisspeptin-IR was characterized in human plasma from patients with malignant GTN

(n=4).

Peptide extraction procedure

Peptide was extracted from plasma using Sep-Pak C18 cartridges (Waters Ltd.,

Hertfordshire, UK) as previously described (34). Briefly, Sep-Pak C-18 cartridges were

activated using 10 ml of 100% methanol and then 20 ml water. Plasma (2ml) from

patients with malignant GTN was mixed with 2 ml of 0.1M HCl and loaded onto the

cartridge. The cartridge was then washed with 10 ml of 4% acetic acid (vol/vol). The Sep-

Pak bound sample was eluted in 1.5 ml methanol and this eluant dried in a Savant

vacuum centrifuge and reconstituted in water plus 0.05% trifluoroacetic acid (TFA)

(vol/vol) for FPLC.

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Reverse phase FPLC

Peptide extracts from plasma were dissolved in 0.6 ml distilled water plus TFA 0.05%

(vol/vol). Of this volume, 0.5 ml was fractionated by FPLC on a high-resolution reverse-

phase (Pep RPC 1 ml HR) C-18 column (Pharmacia, Uppsala, Sweden) as previously

described (1). The column was eluted with a 24.5–29.5% gradient of acetonitrile

(AcN)/water containing 0.05% (vol/vol) TFA over 40 min and fractions collected at 1

min intervals. The kisspeptin-IR in all fractions was determined by radioimmunoassay.

The remaining 0.1 ml of sample was used to calculate the percentage recovery. Recovery

was calculated as kisspeptin-IR (pmol) recovered from each sample, compared with

kisspeptin-IR loaded on to the FPLC column (pmol), multiplied by 100 and expressed as

a percentage. Prolactin releasing peptide was also run on this column to determine its

elution position.

Analytical methods

(i) Human chorionic gonadotrophin, progesterone and oestradiol measurement

Human chorionic gondotrophin was measured by the Endocrine Laboratory at Charing

Cross Hospital (London, UK) using the UK RIA (radioimmunoassay) (6). Progesterone

and oestradiol were measured using commercially available Chemiluminescent

Microparticle Immunoassays (ARCHITECT progesterone and ARCHITECT oestradiol,

Abbott Laboratories, Abbott Park, Illinois, USA).

(ii) Kisspeptin radioimmunoassay (RIA)

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Plasma kisspeptin was measured using an established RIA (9). Briefly, antibody GQ2

was raised in a sheep immunized with synthetic human kisspeptin-54 (Bachem, UK Ltd.),

conjugated to BSA by glutaraldehyde and used at a final dilution of 1: 3,500,000. The

antibody cross reacted 100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10

and less than 0.01% with any other related human RFamide (arginine phenylalanine

amide) peptide, including prolactin releasing peptide, RF amide related peptide (RFRP1),

RFRP2, RFRP 3 neuropeptide FF and neuropeptide AF. The 125I-kisspeptin-54 label was

prepared using the iodogen method and purified by reverse phase high pressure liquid

chromatography on a C18 column (Waters, Milford, USA) over a 15-45% 90 minute

gradient of AcN / water 0.1% (vol/vol) TFA. The specific activity of the kisspeptin label

was 56 Bq/fmol. The assay was performed in duplicate using dilutions of neat plasma in

0.7 ml of 0.06 M phosphate buffer pH 7.2 containing 0.3% bovine serum albumin and

incubated for 3 days at 4oC. Free and antibody bound label were then separated by

charcoal adsorption. The assay detected changes of 2 pmol/L of plasma kisspeptin-IR

with a 95% confidence limit. The intra- and inter-assay coefficients of variation were

8.3% and 10.2% respectively.

Statistical analysis

All results are presented as mean (± SEM). The differences between plasma kisspeptin-

IR pre chemotherapy and post chemotherapy and hCG pre chemotherapy and post

chemotherapy were calculated by a Mann Whitney Rank Sum test. Correlations were

estimated using Pearson Product Moment analysis. In all cases p < 0.05 was considered

to be statistically significant.

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Results

(i) Determination of plasma kisspeptin-IR in healthy female volunteers

Plasma kisspeptin-IR concentration in all non-pregnant females investigated was below

the detection limit of the assay (<2 pmol/L). The plasma kisspeptin in pregnant females

in the first trimester of normal pregnancy (n=13) was 803 ± 125 pmol/L and plasma hCG

was 72,053 ± 10, 936 U/L. The plasma kisspeptin in pregnant females third trimestser of

normal pregnancy (n=7) was 2483 ± 302 pmol/L and plasma hCG was 28,818 ± 11, 348

U/L. By day 15 post partum plasma (n=7), plasma kisspeptin was below the detection

limit of the assay (<2 pmol/L) (Figure 1).

(ii) Determination of plasma hormones in patients at diagnosis of malignant GTN,

during treatment and post chemotherapy

Plasma kisspeptin-IR in patients with malignant GTN were elevated at presentation and

fell during and following treatment with chemotherapy in each patient (plasma

kisspeptin-IR: pre chemotherapy 1363 ± 1076 pmol/L vs. post chemotherapy <2 pmol/L,

n=11, p<0.0001) (Figure 2a, supplementary information Table 1). As expected plasma

hCG levels also showed a similar pattern (plasma hCG: pre-chemotherapy 227191 ±

152354 U/L vs. post chemotherapy 2 U/L, n=11, p<0.0001) (Figure 2b, supplementary

information Table 1). Plasma kisspeptin-IR strongly positively correlated with plasma

hCG levels (r2= 0.99, n=39, p<0.0001, Figure 3). Plasma kisspeptin-IR showed

significant positive correlations with circulating levels of progesterone (r2 = 0.92, n=39,

p<0.0001) and oestradiol (r2 = 0.70, n=39, p<0.0001) as did hCG (r2 = 0.89, n=39,

p<0.0001 for progesterone and r2 = 0.64, n=39, p<0.0001 for oestradiol).

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(iii) Analysis of kisspeptin-IR in human plasma

Reverse-phase FPLC was used to further analyze kisspeptin-IR extracted from plasma by

Sep-Pak cartridge. All columns had a recovery greater than 60%. In each plasma extract

the kisspeptin-IR eluted in a single peak corresponding to the elution position of synthetic

kisspeptin-54 (fractions 14-17). Synthetic prolactin releasing peptide eluted at an earlier

fraction compared to kisspeptin-54 (fraction 10). A representative profile from a patient

with malignant GTN is shown in Figure 4.

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Discussion

Plasma kisspeptin has been proposed as a novel tumor marker indicating the metastatic

potential of specific tumors (14). However, to date no reports have investigated this

possibility by examining the correlation between circulating kisspeptin concentrations

and malignancy. The data described in this paper demonstrate that plasma kisspeptin-IR

is raised in patients with malignant GTN, and falls during treatment. Our results suggest

that plasma kisspeptin-IR may be a novel tumor marker in patients with this disease.

To ensure that the elevated kisspeptin-IR levels observed were not due to levels of

circulating kisspeptin persisting after pregnancy, we compared plasma kisspeptin levels

from malignant GTN patients to those of normal post-partum women. Blood was

collected from GTN patients at first presentation between 1 and 6 months after removal

of their molar pregnancy. Plasma kisspeptin levels in normal women were below the

detection limit of the assay (<2 pmol/L) as early as day 15 post partum, suggesting that

the elevated kisspeptin levels observed in GTN patients were not due to normal post-

partum processes.

Though the kisspeptin/GPR54 system has recently been shown to be critical to normal

reproductive development in rodents and man (7, 12, 39, 40), the role of circulating

kisspeptin is currently unclear. Kisspeptin circulates in non-pregnant females at

concentrations lower than 2pmol/L (14). However, high levels of plasma kisspeptin-IR

are seen in normal pregnancy. The plasma kisspeptin-IR in pregnant females measured

using our single site radioimmunoassay are lower than previously published plasma

kisspeptin levels using a two site enzyme immunoassay (14). However, in accord with

previously published results, plasma kisspeptin was found to be greatly increased in

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pregnancy, and to rise between the first and third trimesters. Further experiments are

needed to determine whether the differences in absolute concentrations detected are due

to, for example, the different assay methods used, the subtle differences in subject

populations, or the slightly different protocols used for blood sampling and storage. There

is currently no international standard protocol for measuring plasma kisspeptin, and it

would be interesting to directly compare the kisspeptin assays currently available.

The source of circulating kisspeptin in pregnant women is assumed to be the placenta,

where the KiSS-1 gene is highly expressed (14, 18). The role of elevated plasma

kisspeptin-IR in pregnancy and malignant GTN is currently unclear, and the release of

kisspeptin may be regulated by different mechanisms in these conditions. Kisspeptin

mediates its effects on the HPG axis via an increase in GnRH. It is therefore possible that

elevated plasma kisspeptin levels in pregnancy chronically increase GnRH release,

leading to a downregulation of the HPG axis, as is seen following the administration of

synthetic GnRH agonists (11). The KiSS-1 gene is an anti-metastasis gene, and

transfection of melanoma cells with KiSS-1 cDNA expression-dependently suppresses

metastasis (21, 23). Kisspeptin-10 has been shown to inhibit trophoblast migration

without affecting proliferation in vitro (1). Interestingly, placental KiSS-1 gene

expression has been shown to inversely correlate with the metastatic potential of patients

with GTN. Placental KiSS-1 gene expression in molar pregnancy is elevated to a similar

degree to that seen in normal pregnancy, but is undetectable in choriocarcinoma (18, 35).

Plasma kisspeptin-IR may therefore be elevated in patients with an invasive mole to

inhibit tumor metastasis and the development of choriocarcinoma (18, 35). Further

studies are required to determine whether plasma kisspeptin-IR is significantly lower in

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patients with choriocarcinoma compared to plasma kisspeptin-IR in patients with molar

pregnancy. It would be of particular interest to investigate whether patients with molar

pregnancy who go on to develop choriocarcinoma have different circulating kisspeptin

levels to those who do not.

Measurement of placental KiSS-1 gene expression requires a placental biopsy, which is

impractical in clinical practice. If plasma kisspeptin acts as a marker of tumor activity, its

measurement would be more clinically applicable. We found large variations in

circulating hCG and kisspeptin concentrations between GTN patients before treatment.

However, plasma hCG and kisspeptin levels showed a strong correlation, and each

patient with a plasma hCG level above the normal range (>5U/L), suggesting disease

activity, also had an elevated plasma kisspeptin. As plasma hCG is one of the factors

used to score disease severity in patients with GTN by the FIGO scoring system (16), this

variation in pre-chemotherapy plasma kisspeptin levels may also reflect differences in

disease activity between subjects.

GTN is currently diagnosed by measuring hCG. Circulating levels of hCG were higher

than those of kisspeptin in the GTN patients in this study. Further work using a larger

group of GTN patients would be required to confirm the sensitivity of hCG compared to

kisspeptin. However, there are problems with using hCG as a diagnostic tool. Though

hCG is a sensitive diagnostic tumor marker, in addition to regular hCG, at least five

major variants of hCG are present in serum samples. In patients with GTN, these variants

of hCG can be the principal circulating form of hCG (4, 5). Some hCG assays do not

detect these hCG variants which can result in a failure to diagnose GTN (5, 19). In

addition, false positive hCG-IR (phantom hCG) results can also occur with hCG assays

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due to proteolytic enzymes that mimic hCG or heterophile antibodies in the serum that

interfere with the hCG assay (3). This can lead to an incorrect diagnosis of GTN and

unnecessary testing and treatment (10). New tumor markers may help to reduce these

problems. Interestingly, though endogenous forms of kisspeptin 54, 14 and 13 amino

acids in length have been isolated from human placenta (20), chromatographic

characterization of the circulating kisspeptin-IR in GTN patients detected only a single

form eluting at an identical position to synthetic kisspeptin-54. Analysis of plasma from

women with a normal pregnancy also shows only a single peak in the same position (data

not shown). Our results suggest that measurement of plasma kisspeptin-IR might be

useful as a novel alternative or complementary tumor marker in patients with malignant

GTN. Further work is required to determine if measurement of plasma kisspeptin-IR

correlates with disease activity in patients with GTN in whom a variant of hCG is the

major circulating form, or in patients with phantom hCG-IR.

The high plasma kisspeptin-IR concentrations in our patients with malignant GTN may

simply reflect the mass of material of placental origin. Kisspeptin is expressed in

trophoblasts, which are known to secrete hCG, oestradiol and progesterone (2, 14).

Circulating concentrations of both kisspeptin-IR and hCG were positively correlated with

circulating oestradiol and progesterone in patients with malignant GTN. There is also a

positive correlation between progesterone and oestradiol levels with kisspeptin levels in

normal pregnancy (14). This might suggest that the release of kisspeptin is related to the

number or mass of trophoblasts (14). However, there is evidence to suggest that

circulating kisspeptin concentrations are not directly correlated to KiSS-1 expression in

the placenta. Although plasma kisspeptin concentration rises throughout pregnancy and

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peaks at term (14), placental KiSS-1 gene expression is not significantly different

between early and term placentas (18). In rats, KiSS-1 gene expression has been found to

decrease in trophoblasts during placental maturation and gestation (1, 47).

Interestingly, although our results suggest that plasma kisspeptin-IR correlates strongly

and significantly with hCG in patients with malignant GTN, there is no significant

correlation between plasma hCG and plasma kisspeptin concentration in pregnancy (14).

Plasma hCG is markedly elevated in the first trimester of pregnancy, but lower levels are

found in the second and third trimester. In contrast, plasma kisspeptin concentrations are

elevated in the first trimester of pregnancy, and continue to increase to a maximum of

approximately 7000-fold basal levels in the third trimester of pregnancy (14).

In conclusion, we have demonstrated that plasma kisspeptin-IR is raised in patients with

malignant GTN and falls during and following treatment. Kisspeptin-IR strongly and

significantly correlated with plasma hCG levels in these patients. Our results suggest that

plasma kisspeptin-IR may be a novel tumor marker in patients with malignant GTN.

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Acknowledgements

We are very grateful to Becky Maddison and Mandy Donaldson for their technical

assistance with the progesterone and oestradiol measurements. The authors would also

like to thank the Department of Health for their continued support of the Charing Cross

Gestational Trophoblastic Disease Centre.

Grants

W.S.D. is funded by a Department of Health Clinician Scientist Fellowship. K.G.M. is

supported by a Biotechnology and Biological Sciences Research Council New

Investigator Award. O.B.C. is funded by a Wellcome Trust Clinical Training Fellowship.

M.P is supported by the Biotechnology and Biological Sciences Research Council. The

Department of Metabolic Medicine is funded by a Medical Research Council programme

grant.

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Figure Legends

Figure 1: Plasma kisspeptin-IR in normal females.

Non-preg = non-pregnant. This included healthy females who had never previously had a

pregnancy in the follicular and luteal phases of their menstrual cycle (n=11) and also

healthy females who had previous pregnancies in the follicular phase of their menstrual

cycles (n=5). First trim = pregnant females in the first trimester of normal pregnancy

(n=13). Third trim = pregnant females at 38 ± 0.8 weeks gestation of normal pregnancy

(n=7). 15d post = females who were day 15 ± 6 post partum (n=7). Values are mean ±sem except for plasma kisspeptin in non-preg and 15d post females which was below the

detection limit of the assay (<2pmol/L).

Figure 2: Plasma kisspeptin-IR (a) and hCG (b) in patients with malignant gestational

trophoblastic neoplasia at presentation (pre chemotherapy) and following chemotherapy

(post chemotherapy).

The thick black line on each graph represents the mean values. n=11. Plasma kisspeptin-

IR in patients with malignant GTN were elevated at presentation and fell during and

following treatment with chemotherapy in each patient (plasma kisspeptin-IR: pre

chemotherapy 1363 ± 1076 pmol/L vs. post chemotherapy <2 pmol/L, n=11, p<0.0001).

Plasma hCG levels also showed a similar pattern (plasma hCG: pre-chemotherapy

227191 ± 152354 U/L vs. post chemotherapy 2 U/L, n=11, p<0.0001).

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Figure 3: Correlation between plasma kisspeptin-IR and hCG in patients with malignant

gestational trophoblastic neoplasia at presentation, during and following chemotherapy.

Correlation was calculated using Pearson’s Product Moment analysis. r2 = 0.99, n=39, p

< 0.0001.

Figure 4: Representative elution profile of kisspeptin-IR extracted from 2 ml plasma by

Sep-Pak cartridge and fractionated by reverse-phase FPLC from a patient with malignant

gestational trophoblastic neoplasia.

Dotted line indicates % AcN = percentage acetonitrile; the elution position of synthetic

kisspeptin-54 was identical to that of plasma kisspeptin-IR from patients with malignant

GTN (fractions 14-17). Synthetic prolactin releasing peptide eluted as fraction 10.

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Figure 1

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Figure 2 a

Figure 2 b

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Figure 3

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Figure 4

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Patientno

Time Kiss (pmol/L)

hCG (U/L)

Prog (ng/mL)

E2 (pg/mL)

1 pre 295 180 000 12 1 078 1 during 41 6 000 1 82 1 post < 2 2 <1 <28 2 pre 20 41 411 11 217 2 during 7 223 2 289 2 post <2 2 2 383 3 pre 101 89 966 25 1 424 3 during 85 17 908 10 550 3 during 13 83 4 435 3 post <2 2 <1 191 4 pre 66 34 655 9 274 4 during 9 157 <1 226 4 post <2 2 <1 170 5 pre 93 18 240 19 277 5 during 5 18 <1 249 5 post <2 2 <1 110 6 pre 37 102 901 9 328 6 during 11 13 <1 <28 6 post <2 2 <1 <28 7 pre 40 21 128 33 3 435 7 post <2 2 <1 337 8 pre 43 20 052 49 3 053 8 during 30 13 232 65 5 918 8 post <2 2 <1 169 9 pre 19 6 453 4 274 9 during 4 50 <1 112 9 post <2 2 <1 <28 10 pre 2 358 251 499 89 9 697 10 during 1 967 226 685 96 12 93610 during 1 945 203 863 79 10 97010 during 984 67 890 61 3 041 10 during 193 5 095 26 2 043 10 during 5 26 <1 <28 10 post <2 3 3 451 11 pre 11 927 1 732 800 243 1 276 11 during 1 764 224 977 67 8 88211 during 203 4 428 8.9 698 11 post <2 4 <1 45

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Supplementary information Table 1: Results of plasma kisspeptin-IR concentration

(Kiss), hCG, progesterone (Prog) and oestradiol (E2) in patients with malignant

gestational trophoblastic neoplasia at presentation (pre), during chemotherapy

(during) and following chemotherapy (post).

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