1411
Metastatic Trophoblastic Disease after an Initial
Diagnosis of Partial Hydatidiform Mole
Genotyping and Chromosome In Situ Hybridization Analysis
Annie N. Y. Cheung, M.D.1
Ui Soon Khoo, M.D.1
Caroline Y. L. Lai, M.Med.Sc.1
Kelvin Y. K. Chan, Ph.D.1,2
Wei-Cheng Xue, M.D.1
Danny K. L. Cheng, M.D.2
Pui-Man Chiu, M.Med.Sc.1
Sai-Wah Tsao, Ph.D.3
Hextan Y. S. Ngan, M.D.2
1
Department of Pathology, The University of Hong
Kong, Hong Kong, China.
2
Department of Obstetrics and Gynecology, The
University of Hong Kong, Hong Kong, China.
3
Department of Anatomy, The University of Hong
Kong, Hong Kong, China.
Supported by grants from the Research Grant
Council of Hong Kong.
The authors thank Mr. Tony Chan for his technical
advice.
Address for reprints: Annie N. Y. Cheung, M.D.,
Department of Pathology, The University of Hong
Kong, Queen Mary Hospital, Pokfulam Road, Hong
Kong, China; Fax: (011) 852 28725197; E-mail:
[email protected]
Received October 27, 2003; revision received December 21, 2003; accepted January 6, 2003.
© 2004 American Cancer Society
DOI 10.1002/cncr.20107
BACKGROUND. Hydatidiform mole (HM) is classified into partial (PHM) and complete (CHM) subtypes according to histopathologic and genetic criteria. Traditionally, it is believed that PHM carries a better prognosis and rarely develops metastasis. However, making a distinction between PHM and CHM using histologic
criteria alone may be difficult.
METHODS. The authors used fluorescent microsatellite genotyping following lasercapture microdissection and chromosome in situ hybridization (CISH) to perform
a genetic analysis of six patients with histologically diagnosed PHM who subsequently developed metastatic gestational trophoblastic neoplasia.
RESULTS. Patients ranged in age from 25 years to 44 years (mean, 33.2 years). The
gestational age of the molar pregnancies varied from 6 weeks to 20 weeks. All six
patients had pulmonary metastases, with additional liver metastasis in two patients. Among the six patients with histologically diagnosed PHM, it was found that
four patients had a diploid karyotype and no maternal alleles; thus, their neoplasms actually were CHM. Maternal genome was detected in the remaining two
patients consistent with a biparental origin, and these patients had a triploid
karyotype. CISH findings in all patients correlated with the genotyping findings.
Triploid HM had maternally derived alleles, whereas diploid HMs were purely
androgenetic.
CONCLUSIONS. In the current study, which may be the largest series of genetically
analyzed metastatic PHMs to date, the difficulty of histologic distinction between
PHM and CHM was confirmed. Molecular analysis may help to refine the classification of HM. Although the current findings support the belief that most aggressive trophoblastic diseases are derived from CHM, a small number of PHMs do
progress to metastatic disease. Thus, the current study reaffirmed that all patients
with HM should be followed closely irrespective of histologic subclassification.
Cancer 2004;100:1411–7. © 2004 American Cancer Society.
KEYWORDS: metastatic partial mole, genotyping, chromosome in situ hybridization,
gestational trophoblastic disease.
G
estational trophoblastic disease (GTD) is a disease of the trophoblastic tissue that includes a heterogeneous variety of lesions with variable degrees of neoplastic changes. GTD includes benign hydatidiform
mole (HM), invasive mole, choriocarcinoma, placental site trophoblastic
tumor, and epithelioid trophoblastic tumor.1,2 HM is the most common
type and is characterized by significant hydropic enlargement and variable trophoblastic hyperplasia. Although the majority of HMs spontaneously regress after suction evacuation, some may develop gestational
trophoblastic neoplasia (GTN) and thus require chemotherapy. Metastases may develop occasionally. HM can be subclassified into complete
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CANCER April 1, 2004 / Volume 100 / Number 7
TABLE 1
Correlation of Clinical Parameters, Histologic Diagnosis, and Findings on Genotyping and Ploidy Analysis
Patient
no.
Patient
age
(yrs)
47
49
50
52
54
55
36
44
31
35
25
28
Wks of
gestation
Initial
pathologic
diagnosis
Presence
of fetal
tissue
Mos
between
diagnosis
and CT
Treatment
risk factor
Site(s) of
metastases
Mos
between
diagnosis
and
metastasis
detection
11
10
20
10
6
9
PHM
PHM
PHM
PHM
PHM
PHM
No
No
No
No
No
No
9
2
2
3
2
3
M
H
H
M
M
M
Lung
Lung, liver
Lung, liver
Lung
Lung
Lung
0
0
0
0
4
0
Presence of
maternal
genome
(genotyping)
Ploidy
(CISH)
Reviewed
pathologic
diagnosis
No
No
Yes
Yes
No
No
Diploid
Diploid
Triploid
Triploid
Diploid
Diploid
CHM
CHM
PHM
PHM
CHM
CHM
CT: chemotherapy; CISH: chromosome in situ hybridization; PHM: partial hydatidiform mole; CHM: complete hydatidiform mole; M: medium risk; H: high risk.
(CHM) and partial (PHM) subtypes based on morphologic, pathologic, and genetic differences.3–5 In general, it
is believed that CHM carries a much greater risk of
developing GTN. Nevertheless, PHMs with aggressive
sequelae occasionally are reported.6 –10
Although CHM and PHM traditionally are considered to be histologically distinguishable, the diagnosis
of HM based on morphologic differences alone has
the problem of interobserver and intraobserver variability.11–16 Techniques that make use of the genetic
difference between PHM and CHM have been developed to assist in making the differential diagnosis.
Genetically, CHMs usually are diploid and androgenetic in origin, with all 46 chromosomes originating
entirely from the sperm.17–19 In contrast, most PHMs
are triploid and are formed by dispermic fertilization
of a normal ovum.20,21 Thus, both PHMs and CHMs
possess two sets of paternal haplotypes (diandric) in
the genome. Nevertheless, unlike CHMs, which have
no maternal contribution, PHMs have one set of chromosomes contributed by the maternal ovum.
In the current study, retrospective genetic analysis
was performed for six patients with rare cases of histologically diagnosed PHM from which metastatic disease subsequently developed. To our knowledge, it is
likely that the current study is the largest series to date
involving genetic analysis of metastatic PHMs. We
used fluorescent microsatellite genotyping to identify
the parental origin of the molar tissue; this technique,
together with chromosome in situ hybridization
(CISH),22 can be used to identify genuine metastatic
PHMs.
MATERIALS AND METHODS
Patients
Patients with histologically diagnosed PHM who subsequently developed metastatic GTN were identified
from the clinical records of Department of Obstetrics
and Gynecology and the Department of Pathology at
Queen Mary Hospital, The University of Hong Kong
(Hong Kong, China), between 1989 and 1996. After
suction evacuation of the molar pregnancy, patients
were monitored in a standard fashion, including serial
serum and urinary human chorionic gonadotrophin
(␤-hCG) assays.23 In our center, GTN was suspected
when ␤-hCG levels remained the same for 4 weeks or
if there was a rising ␤-hCG level for 3 consecutive
weeks when pregnancy was excluded. Patients with
suspected GTN were evaluated for evidence of metastatic disease.
Most patients with metastatic PHM in the current
study were referred to our institution. The diagnosis of
metastatic trophoblastic disease was based on clinical,
serologic, and radiologic evidence. No tissue diagnosis
of the metastatic lesion had been obtained. The original pathologic diagnosis had been made based on
histologic criteria on hematoxylin and eosin (H&E)–
stained sections from each patient.1,2 Partial mole was
diagnosed when there was a certain degree of fetal
development, including nucleated red blood cells, or
when the hydropic change was observed in only a
portion of the chorionic villi. Trophoblastic proliferation usually was focal.
Formalin-fixed, paraffin-embedded tissue samples containing both maternal endometrium and molar villi were obtained successfully by uterine curettage in six patients. The clinical and pathologic
features of all patients are listed in Table 1. Patients
ranged in age from 25 years to 44 years (mean, 33.2
years). The gestational age of the molar pregnancies
varied from 6 weeks to 20 weeks. All patients had
serum hCG levels that remained stationary for at least
4 weeks. Investigations included chest X-ray, pelvic
and hepatic arteriograms, and magnetic resonance
Genotyping of Metastatic Partial Mole/Cheung et al.
1413
TABLE 2
Microsatellite Primers
Locusa
Location
Repeat
Heterozygosity
Primer sequence
D13S267
13q12
Diploid
0.69
D17S1322
17q21
Diploid
0.67
D17S855
17q21
Diploid
0.82
D5S818
5q22
Tetraploid
0.74
D3S1358
3p21
Tetraploid
0.78
vWA
12p13–pter
Tetraploid
0.80
5⬘-GGCCTGAAAGGTATCCTC-3⬘
5⬘-TCCCACCATAAGCACAAG-3⬘
5⬘-CTAGCCTGGGCAACAAACGA-3⬘
5⬘-GCAGGAAGCAGGAATGGAAC-3⬘
5⬘-GGATGGCCTTTTAGAAAGTGG-3⬘
5⬘-ACACAGACTTGTCCTACTGCC-3⬘
5⬘-GGGTGATTTTCCTCTTTGGT-3⬘
5⬘-TGATTCCAATCATAGCCACA-3⬘
5⬘-ACTGCAGTCCAATCTGGGT-3⬘
5⬘-ATGAAATCAACAGAGGCTTG-3⬘
5⬘-CCCTAGTGGATAAGAATAATC-3⬘
5⬘-GGACAGATGATAAATACATAGGATGGATGG-3⬘
a
References of the loci were obtained from the Genome Data Base (http:///www.gdb.org).25
images of the brain for patients with lung metastasis.
All patients had lung metastasis that varied in size
from 5.0 mm to 1.5 cm. Two patients had liver metastasis, as evidenced on hepatic arteriograms, that
ranged in size from 6 mm to 10 mm. Two patients
were treated with methotrexate, two patients were
treated with actinomycin-D and methotrexate, and
two patients were treated with modified hydroxurea,
vincristine, methotrexate, actinomycin-D, and cyclophosphamide chemotherapy regimens. All patients responded well to chemotherapy and remained free of
disease for 8 –14 years.
DNA Preparation
For each specimen, the maternal decidua and molar
villi were microdissected separately from 6 ␮m, H &
E–stained sections using the Arcturus PixCell威 II LM
200 Laser Capture Microdissection (LCM) System
(Arcturus Engineering, Mountain View, CA).22 DNA
then was prepared from the laser microdissected tissue according to the manufacturer’s protocol.24 In
brief, DNA from the dissected cells on the Capsure™
HS LCM cap (Arcturus Engineering) was extracted by
bringing the cap surface into contact with 50 ␮L digestion buffer containing 0.04% proteinase K; 10 mM
Tris-HCl, pH 8.0; 1 mM ethylenediamine tetraacetic
acid (EDTA); and 1% Tween 20 (Sigma Chemical Co.,
St Louis, MO). After overnight incubation at 37 °C, the
extraction tube was centrifuged, and the fluid was
heated to 95 °C for 10 minutes to inactivate the proteinase K.
Polymerase Chain Reaction
In each case, 3 ␮L of DNA from both maternal and
molar tissue samples were amplified with 6 pairs of
primers that flanked polymorphic microsatellite re-
peat sequences on 5 different chromosomes (Table
2).22 The loci included D3S1358, D5S818, D13S267,
D17S1322, D17S855, and vWA.25 Polymerase chain reaction (PCR) analysis was performed in a 10 ␮L reaction volume containing 1X PCR buffer (10 mM TrisHCl and 50 mM KCl, pH 8.3), 2.0 mM or 2.5 mM
magnesium chloride, 250 ␮M of each deoxyribonucleoside triphosphate, 0.4 ␮M each of the forward
and reverse primers, and 0.6 units of AmpliTaq Gold威
(Applied Biosystems, Foster City, CA). One primer
from each primer pair was labeled with a fluorescent
dye—fluorescein, hexachlorofluorescein, or carboxytetramethylrhodamine. The PCR reaction consisted of an initial denaturation step at 95 °C for 12
minutes; followed by 39 cycles of denaturation at 94 °C
for 30 seconds, annealing at 60 °C for 1 minute, with
extension at 72 °C for 40 seconds; and a final extension
at 72 °C for 10 minutes using the GeneAmp威 PCR
System 9600 (Applied Biosystems). After amplification, 2.5 ␮L of each PCR reaction product underwent
electrophoresis in a 2% agarose gel to assess the yield.
Microsatellite Genotyping
The PCR products from each sample were then diluted
according to the yield. After diluting with an appropriate volume of a mixture of formamide and blue
dextran/EDTA containing a carboxy-x-rhodamine-labeled internal size standard, the PCR products were
denatured at 95 °C, placed on ice to chill, and then
separated by electrophoresis in a 5% denaturing polyacrylamide gel using the ABI PRISM 377 DNA Sequencer (Applied Biosystems).22 Data analysis and
fragment sizing of the microsatellite polymorphism
were performed using ABI PRISM GeneScan威 analysis
software (Version 3.1; Applied Biosystems) and Genotyper威 fragment analysis software (Version 2.5; Ap-
1414
CANCER April 1, 2004 / Volume 100 / Number 7
plied Biosystems). The allele sizes of microsatellite
polymorphisms found in the molar villi then were
compared with the corresponding allele sizes observed in the maternal tissue.
CISH
The ploidy of each HM was studied with CISH.23,26 –29
DNA probes specific for the pericentromeric regions
of chromosome 11 (D11Z1), chromosome 16 (D16Z1),
chromosome Y, DYZ5 (American Type Culture Collection, Rockville, MD), and chromosome X (pBamX5; a
generous gift from Dr. A. H. N. Hopman; Department
of Molecular Cellular Biology and Genetics, University
of Limburg, Maastricht, The Netherlands) were
used.30,31 All DNA probes were labeled with biotin
11-deoxyuridine triphosphate using the standard
nick-translation method (Boehringer Mannheim, Indianapolis, IN).
Paraffin sections (thickness, 5 ␮m) were mounted
on 2% aminopropyltriethoxysilane-coated slides. After
deparaffinization, the slides were incubated with proteinase K (250 –500 ␮g/mL) and 0.1% Triton X-100 at
50 °C. The labeled probes were added to the hybridization mix (60% formamide, 10% dextran sulfate, 2
⫻ standard saline citrate) and applied to the tissue
sections at probe concentrations of 1 ng/␮L of hybridization mixture. Denaturation was performed at 90 °C,
followed by overnight hybridization at 37 °C with intermittent agitation. Immunohistochemical analysis
was performed using avidin, biotinylated mouse antiavidin, rabbit antimouse peroxidase, and 3,3⬘-diaminobenzidine (Dakopatts Ltd., High Wycombe, United
Kingdom). Hydrogen peroxidase was used to visualize
peroxidase activity.
In each of the sections that was hybridized with
individual, chromosome-specific DNA probes, at least
200 nuclei were assessed by independent observers
(W.-C.X. and A.N.Y.C.). A gain in chromosome 11 or
chromosome 16 was inferred when ⱖ 15% of the tumor cells displayed 3 signals or ⬎ 3 signals.
FIGURE 1. Microsatellite polymorphisms and the diploid hydatidiform mole
(HM) in Patient 46. Patient 46 was homozygous for the marker D17S1322,
generating an allele of 117 base pairs. The HM also was homozygous for
D17S1322, giving rise to an allele of 123 base pairs.
contribution (Fig. 1), while a maternal contribution
was observed in two moles (Patients 50 and 52).
CISH
From CISH studies, triploidy was inferred in two
moles (Patients 50 and 52), whereas the other four
moles (Patients 47, 49, 54 and 55) exhibited a diploid
composition (Fig. 2). No signal for the Y chromosome was documented in any of the six moles. In all
moles, genotyping findings were correlated with
CISH findings. Triploid HMs had maternally derived
alleles, whereas diploid HMs were purely androgenetic (Table 1).
RESULTS
Fluorescent Microsatellite Genotyping
Histology
In the interpretation of genotyping results, the absence of maternal genome in the HM was indicated by
the exclusion of maternal allele(s) in the molar tissues.
Alleles in the molar tissue that were not found in the
maternal tissue were interpreted as being paternal. In
contrast, HMs with one or more maternal alleles definitively demonstrated by microsatellite markers were
interpreted as being biparental. Four of six metastatic
moles (Patients 47, 49, 54 and 55) exhibited genotyping profiles that indicated an absence of maternal
Histologic review, which included the two genetically
biparental moles (Patients 50 and 52), did not reveal
fetal tissue or nucleated red blood cells. The presence
of two groups of chorionic villi (one group was more
hydropic) was more conspicuous in the two true
PHMs.
Correlation between Histologic Diagnosis and Genotyping
Genotyping and CISH results were correlated with
histology in 2 of 6 patients (33.3%) (Table 1). For all
Genotyping of Metastatic Partial Mole/Cheung et al.
FIGURE 2. Two hybridization signals, as detected by DNA probes for chromosome 16, were found in the majority of nuclei in the mole from Patient 46.
patients, genotyping findings agreed with CISH findings.
DISCUSSION
PHM may be underdiagnosed as abortion; it also may
be diagnosed as CHM. The diagnosis of CHM is particularly difficult in patients with early evacuated
moles, in which the distinct features of villous edema
and trophoblastic hyperplasia may not have developed fully. Most moles that are diagnosed initially as
PHM, including the HMs in the current study, are
diagnosed before 10 weeks. This may explain why the
full-blown histologic features of CHM were not conspicuous. It has been reported that in early evacuated
CHMs (during the first 5–9 weeks of gestation), in
which the distinct features of villous edema and trophoblastic hyperplasia are not fully developed, histologic distinction between CHM and PHM may be particularly difficult. CHM may be misdiagnosed as PHM
or even as hydropic abortion.11–14,32–34 PHMs also are
quite easily misdiagnosed as CHMs.30 Previous studies
demonstrated that most persistent trophoblastic diseases that resulted from histologically diagnosed PHM
were in fact CHM according to their diploid DNA
patterns.31 Even the presence of amnion or other fetal
tissues associated with molar tissue cannot always be
considered indicative of a diagnosis of PHM, because
these tissues may be androgentic in origin and may
indicate the presence of early embryonic development
in CHM.35
Although it is uncommon, PHM also can lead to
persistent disease6,8,9,10,36 or even choriocarcinoma.7
In the study conducted by Seckl et al.,7 for example, of
the 3000 patients with PHM, only 15 required chemo-
1415
therapy for persistent disease. The development of
metastatic disease in patients with PHM is even more
unusual.
Because CHM and PHM genetically are distinct,
several techniques that make use of these differences
have been developed to help improve the accuracy of
diagnosis. Cytogenetic analysis, DNA flow cytometry,
and CISH are useful in determining the ploidy of the
molar tissue. CHMs usually are diploid, whereas most
PHMs are triploid. However, cytogenetic analysis by
karyotyping requires fresh tissue for cell culture and
preparation of metaphase spreads.
When fresh tissue is not available for karyotyping,
ploidy determination can be achieved by DNA flow
cytometry and CISH. Both methods can be applied to
fixed, paraffin embedded tissues. Using flow cytometry, the large difference of diploid and triploid cell
populations, such as in CHM and PHM, can be detected easily.37– 41 CISH also aids in the classification of
HM by determining ploidy patterns, differentiating
between XX and XY CHMs, and deducing the paternal
contribution in CHMs using DNA probes specific for
the Y chromosome.25,41,42
PCR aids in the rapid diagnosis and typing of
HMs.43– 47 Recently, fluorescent-labeled microsatellite
primers were used in the genotyping of HMs.22 Using
this technique, samples can be run on and analyzed
by an automated DNA sequencer, thus enabling quick
analyses of large numbers of DNA polymorphisms on
different samples at one time.
It is likely that the current study represents the
largest series of genetically analyzed metastatic PHMs.
We conclude that genotyping using microsatellite
analysis and CISH can reliably ascertain the histologic
diagnosis and subclassification of HM using formalinfixed, paraffin embedded tissues of molar villi and
decidua, even when paternal tissue is not available for
evaluation. Furthermore, this method allows retrospective analysis of archival material. Therefore, this
approach is useful in routine surgical pathology reporting. Our findings demonstrate that in some cases,
aggressive disease resulting from histologically diagnosed PHM may in fact be CHM. However, a small
number of PHMs did progress to metastatic disease.
Because it is difficult to distinguish PHM from CHM
histologically, all patients with HM should be followed
closely.
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