Gestational Trophoblastic Disease
Moderator: Dr. le-Ming Shih
Section 6 -
Application of New Laboratory Techniques in Diagnosis and Prognosis Prediction of Molar Lesions
Annie NY Cheung
Department of Pathology, Queen Mary Hospital
The University of Hong Kong
In recent years, advances in laboratory techniques have enabled more elaborate biological studies of
gestational trophoblastic disease  . It has been highlighted that histological distinction between
hydropic abortion and partial mole and between complete and partial moles, especially at early
gestational age, may be difficult. Moreover, few parameters have been found useful in predicting the
clinical progress of hydatidiform mole to persistent gestational trophoblastic neoplasia which requires
chemotherapy  . Similar to other human cancers, malignant transformation in gestational trophoblastic
tumors is likely a multistep process involving activation of oncogenes and inactivation of tumor
suppressor genes. In addition, disruption of the delicate regulation of cellular processes including
proliferation, differentiation, apoptosis and invasion, as well as expression of telomerase activity, may
also be important mechanisms in determining behavior of these diseases. The application of these
techniques on such processes as attempts to improve diagnosis and management of gestational trophoblastic
disease will be discussed.
Hydatidiform mole is classified into partial and complete subtypes according to histopathological and
. Genetically, a complete mole is diploid without maternal contribution, whereas
a partial mole is triploid with a maternal chromosome complement
 . Various diagnostic tools have been considered useful for such distinction including cytogenetic
analysis, DNA flow cytometry, chromosome in situ hybridization and PCR based genotyping
Fluorescent microsatellite genotyping to detect the presence or absence of maternal
genome in a hydatidiform mole and chromosome in situ hybridization to analyze the ploidy have been
performed to correlate with histological diagnosis  . The genotyping results correlated with
chromosome in situ hybridization findings in all cases, i.e. triploid hydatidiform moles had
maternal-derived alleles, while diploid hydatidiform moles were purely androgenetic. Compared with
genetic diagnosis, histological evaluation was more reliable for the diagnosis of a complete mole than
that of a partial mole. Genotyping and chromosome in situ hybridization can thus provide reliable
adjunct to histology for the classification of a hydatidiform mole.
As a partial
mole still carries a risk of developing gestational trophoblastic neoplasia, follow-up is considered
necessary for both complete and partial moles. Actually, among the six histologically diagnosed partial
hydatidiform mole which subsequently metastasized, four cases had a diploid karyotype and no maternal
alleles. These cases were thus actually partial hydatidiform mole  . However, it seems that there
was no correlation between the presence of a Y chromosome and the development of persistent gestational
trophoblastic disease with or without metastasis
Telomerase is a ribonucleoprotein complex that is thought to add telomeric DNA repeats onto the ends
of chromosomes during cell proliferation. Since cells of most normal tissues lack this activity,
successive cell division will result in progressive shortening of telomeres. It is hypothesised that
telomere shortening both in vivo and in vitro
is the mitotic clock that determines the onset of cellular senescence. It is also suggested that
activation of telomerase is necessary for the sustained proliferation and development of tumours
. Telomerase activity in hydatidiform mole was assessed and compared with normal placentas of different
gestational age and choriocarcinoma using the telomeric repeat amplification protocol (TRAP) assay.
Telomerase activation was demonstrated in hydatidiform mole. Hydatidiform mole, which subsequently
developed persistent disease, especially those which metastasised, were more likely to express telomerase
activity  . Thus, assessment of telomerase activity in hydatidiform mole may be useful in the
Proliferative and Apoptotic Activities
The role of assessing proliferative activity by immunohistochemistry for the proliferating cell
nuclear antigen (PCNA), Ki-67, m inichromosome maintenance protein mcm7 and helix-loop-helix Id proteins
(inhibitor of differentiation or inhibitor of DNA binding) , as a measure to predict the clinical
progress of hydatidiform mole have been assessed. There was no statistically significant difference in
such proliferative index between those patients who developed persistent disease and those who did not
. The proliferation index is probably not useful in predicting the prognosis of molar
pregnancies. On the other hand, proliferation index assessed by the newer generation of cell cycle
markers such as MCM7 and Id was significantly higher in partial hydatidiform mole and complete
hydatidiform mole than in spontaneous abortion and may thus be useful to distinguish hydropic abortion
from early hydatidiform mole
More promising results were obtained from studies on apoptotic activity in GTD. The apoptotic
activity was initially studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine
triphosphate (dUTP) nick end labeling (TUNEL)
method  . Apoptotic activity were significantly
different among normal placenta, spontaneous abortion, choriocarcinoma and hydatidiform mole. Apoptotic
indices of those hydatidiform moles that spontaneously regressed was statistically higher than those that
developed persistent gestational trophoblastic neoplasia. A poptotic activity of trophoblastic lesions
in general inversely correlated with Bcl-2 expression but no t Bax expression. A poptotic index may thus be a useful prognostic marker for
clinical progress of hydatidiform mole . Bcl-2 expression is probably
regulating apoptosis in trophoblastic lesions while Bax expression is not.
Such findings were further confirmed when apoptotic activity of gestational trophoblastic disease was
further assessed using the M30 CytoDeath antibody  . Apoptotic indices assessed by M30 antibody and
TUNEL correlated with each other.
Both the TUNEL method and M30 immunohistochemistry are techniques that can be conveniently applied in
routine surgical pathology and provide useful information for prediction of clinical progress of
Differential Expression Display Studies
Recent advances in molecular methodology have facilitated high-throughput analysis of differential
gene expression in GTD. cDNA array is available from commercial companies and human genome centers
allowing simultaneous analysis of various genes known to be involved in human carcinogenesis, cell cycle
and transcription regulation, DNA repair and synthesis, apoptosis, growth factors and receptor signaling
 . The differential expression profile of genes with known functions can then be identified leading
to better understanding of the disease  . On the other hand, Suppression Subtractive Hybridization
(SSH) technique, which combines both the suppression PCR amplification and cDNA subtraction, is sensitive
and efficient for generating cDNA highly enriched for differentially expressed genes with both high and
low abundance leading to generation of two subtractive libraries  . These may include novel
transcripts with uncharacterized functions.
These approaches have been utilized
to study gestational trophoblastic disease from two angles. Firstly, differentially expressed genes can
be identified in hydatidiform mole related to different clinical outcome, i.e. spontaneous regression and
developing persistent gestational trophoblastic neoplasia . Such genes may be useful marker for
predicting progress of hydatidiform mole . Secondly, differentially expressed genes in hydatidiform mole
as compared with non-molar placentas can be identified and these genes may contribute toward
understanding the pathogenesis of complete hydatidiform mole and may be explored as markers for diagnosis
of hydatidiform mole in histologically difficult cases.
HM That Developed GTN Vs Spontaneously Regressed HM
Due to earlier findings that low apoptotic activity
correlated with the progression of hydatidiform mole to gestational trophoblastic neoplasia ,
apoptosis-related genes may determine this progression. The differential expression of apoptotic genes
in hydatidiform mole s that subsequently developed into gestational trophoblastic neoplasia was compared
with hydatidiform mole s that spontaneously regressed using a human apoptosis array. Increased
expression of Mcl-1, an antiapoptotic gene, was detected in hydatidiform mole s that subsequently
developed into gestational trophoblastic neoplasia
 . It was confirmed by follow up quantitative real time PCR and protein expression analysis.
Moreover, Mcl-1 immunoreactivity, which was detected predominantly in cytotrophoblasts, was correlated
with the apoptotic index, assessed with M30 cytoDeath immunohistochemistry.
Tissue-specific chips were constructed from the subtracted cDNA libraries comparing the differential
expression pattern of genotyped hydatidiform mole that spontaneously regressed and that subsequently
developed metastatic gestational trophoblastic neoplasia , followed by cDNA microarray analysis. Among
the differentially expressed transcripts identified, quantitative RNA analysis confirmed down-regulation
of ferritin light polypeptide (FTL) and insulin-like growth factor binding protein 1 (IGFBP1) in
hydatidiform mole that subsequently developed gestational trophoblastic neoplasia when compared with
those hydatidiform mole that regressed
 . Immunohistochemical analysis further confirmed reduced IGFBP1 protein expression in hydatidiform
mole that developed gestational trophoblastic neoplasia . Our findings suggested that reduced expression
of genes related to cell invasion and immunosuppression, especially FTL and IGFBP1, were associated with
development of gestational trophoblastic neoplasia , and this finding may provide a better understanding
of the pathogenesis of gestational trophoblastic neoplasia . The potential application of FTL and IGFBP1
in management of patients with hydatidiform mole should be explored.
GTD Vs Non-Molar Trophoblasts
Using cDNA array, Vegh et al has demonstrated down-regulation of
Hsp-27 in choriocarcinoma cell lines suggesting that it may contribute to the extreme sensitivity of
trophoblastic tumors to chemotherapy  . Besides confirming such findings, our group employing cDNA
array hybridization to compare gene expression profiles in choriocarcinoma cell lines (JAR, JEG, and
BeWo) and normal first trimester human placentas, has also identified differential expression of Caspase
10 and its closely related family member caspase 8 in choriocarcinoma  . Down-regulation of caspase
10 in choriocarcinoma was detected by both human cDNA expression array and human 1.2 array. Caspase 10
mRNA expression was significantly lower in hydatidiform mole and choriocarcinoma compared with normal
placenta. The caspase 8 and 10 proteins were expressed predominantly in the cytotrophoblast and
syncytiotrophoblast, respectively, with significantly lower expression in choriocarcinomas than other
trophoblastic tissues. Immunoreactivity for both caspase 8 and 10 correlated with the apoptotic index
previously assessed by TUNEL and M30 approaches. These results suggest that the downregulation of
capases 8 and 10 might contribute to the pathogenesis of choriocarcinoma.
attempt to assess the molecular pathogenesis of complete hydatidiform mole , suppression subtractive
hybridization (SSH) combined with cDNA microarray was used to compare the gene expression pattern of
complete hydatidiform mole compared with normal first-trimester placenta of similar gestational ages
 . cDNA microarray analysis using tissue-specific chips constructed with subtracted cDNA libraries
identified 13 differentially expressed gene transcripts. Quantitative real-time PCR confirmed
up-regulation of human chorionic gonadotropin beta subunit (CGB) and KIAA1200, a G-protein regulator, as
well as down-regulation of osteopontin (SPP1) in genotyped complete hydatidiform mole when compared with
normal placentas. Down-regulation of osteopontin has been previously reported by Batorfi et al from a
candidate gene approach  . These candidate genes may contribute toward understanding the mechanism
involved with the development and progression of complete hydatidiform mole .
Epigenetic Downregulation of Tumor Suppressor Genes
While promoter hypermethylation has recently been found to be an important epigenetic mechanism
causing gene inactivation, the methylation status of genes in hydatidiform mole and choriocarcinoma and
its significance is relatively unexplored. The methylation status of the promoter regions of six genes,
p16, HIC-1, TIMP3, GSTP1, death-associated protein kinase (DAPK), and E-cadherin in hydatidiform moles,
choriocarcinomas, and first trimester placenta have recently been studied by methylation-specific PCR in
correlation with protein and quantitative real-time RT-PCR studies. Among the six genes examined, the
promoter region of four genes (E-cadherin, HIC-1, p16, TIMP3) in choriocarcinoma and three genes
(E-cadherin, HIC-1, p16) in hydatidiform mole exhibited aberrant methylation whereas none was
hypermethylated in normal placenta. Treatment with a demethylation drug, 5-aza-2'-deoxycytidine, in
choriocarcinoma cell lines restored TIMP3 expression confirming that promoter methylation of TIMP3 is
involved in suppression of TIMP3 expression.  . There was a significant correlation between
methylation and reduced expression of p16, E-cadherin, and TIMP3. Moreover, promoter hypermethylation of
p16 alone, or combined with E-cadherin, was significantly correlated to development of gestational
trophoblastic neoplasia . Thus, hypermethylation of multiple genes, especially p16, might be related to
the subsequent development of gestational trophoblastic neoplasia  .
With recent advances in molecular techniques, molecular factors in the pathogenesis of gestational
trophoblastic disease have been studied. Yet, further works are still necessary to provide a better
understanding of this peculiar group of disease, which may not only provide important insights into its
pathogenesis, but also useful prognostic indicators which may guide therapy.
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