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2GENETICSRosemary A Fisher

2.1 GENETIC ORIGIN OF GESTATIONALTROPHOBLASTIC DISEASE

Gestational trophoblastic disease (GTD) includes hydatidiform moles (HM),both complete (CHM) and partial (PHM), invasive mole (IM) and themalignant gestational trophoblastic tumours (GTT) choriocarcinoma andplacental site trophoblastic tumour (PSTT). While many genetic studies havebeen performed in both CHM and PHM, similar studies of trophoblastic

tumours have been limited by the availability of fresh material. Theintroduction of molecular techniques, in particular those which can be usedin conjunction with fixed material has facilitated both diagnosis and furtherstudies of the pathogenesis of this unusual group of diseases.

2.1.1 HYDATIDIFORM MOLES

2.1.1.1 Karyotypes of HM

Early genetic studies of HM found that most molar pregnancies were female

in that Barr bodies, darkly staining bodies found in the interphase nuclei offemale cells [1], were present in the majority of HM [2]. Cytogenetic studiesconfirmed this by showing that most HM had a 46,XX female karyotype [3-6]. These studies also revealed a small number of HM with triploidkaryotypes [7,8]. Correlation of the morphology of HM with studies of theirkaryotype led Vassilakos and his colleagues [9,10] to suggest that the twopathological entities CHM and PHM were genetically distinct. Vassilakos etal [9-10] found all CHM to have a diploid 46,XX karyotype while PHM weremainly triploid or trisomic for a single chromosome. PHM were classified asconceptuses with a fetus, cord or amniotic membranes and a placentacomprising both normal and cystic villi. Hyperplasia, present in some cases,

was not marked. When hyperplasia was included as a prerequisite for thepathological diagnosis of PHM [11,12] it became apparent that PHM werealmost always triploid [13-15]. Thus PHM was shown to be a separate entityand not, as had previously been suggested by Hertig and Edmonds [16], atransitional stage between normal placenta and CHM. PHM may bekaryotypically 69,XXX; 69,XXY or 69,XYY [11,17,18].


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2.1.1.2 Parental Origin of CHM

CHM and PHM are genetically distinct. Interestingly it is the CHM, with theapparently normal diploid karyotype, which is the genetically more unusualtissue. Using cytogenetic polymorphisms, ie inherited staining patterns on

chromosomes, two groups demonstrated that CHM were androgenetic inorigin [19,20], all 46 chromosomes in the molar tissue being paternallyderived. Other groups [13,21-24] soon confirmed this surprisingobservation. Early studies using cytogenetic polymorphisms, which arecentromeric markers, found that homologous pairs of chromosome in CHM

were homozygous for all informative markers examined [19,20]. Theobservation that polymorphic enzyme markers, located distally on thechromosome, were also homozygous led to the conclusion that CHM arosefollowing duplication of a haploid sperm rather than fertilization of an eggby a diploid sperm that had failed to undergo reduction during meiosis[13,23,24]. Since CHM with a 46,YY karyotype have not been reported,

duplication of sperm carrying a Y chromosome is presumed to be non-viable.

Duplication of a haploid sperm could not account for the smallproportion of CHM with a 46,XY karyotype [3,6,25]. In 1981 Ohama et aldescribed four androgenetic, 46,XY CHM in which half the informativepolymorphisms studied were homozygous, but half were heterozygous,suggesting that 46,XY CHM arose by dispermy [26]. Several other examplesof both 46,XY and 46,XX CHM arising by dispermy have now beenreported [27-31]. The incidence of dispermic CHM varies in different series.

While 46,XY dispermic CHM can be easily identified by the presence of a Ychromosome, a number of informative polymorphisms may need to be

examined in order to identify heterozygosity in a 46,XX CHM [32]. Wheremore informative markers, such as restriction fragment lengthpolymorphisms of DNA, have been used to examine the origin of CHM, theincidence of dispermic CHM has been shown to be in the region of 20 -25% [18,32,33].

Despite the androgenetic nature of the nuclear genome, analysis of DNApolymorphisms has shown the mitochondrial DNA of CHM, as in a normalconceptus, to be maternally derived [34-36]. These findings suggest thatCHM arise from the fusion of one or two sperm with a mature but anucleateegg. The fate of the maternal chromosomes in CHM is unclear. Extrusion ofboth maternal sets of chromosomes or chromatids into one of the polar

bodies during meiosis could give rise to an anucleate egg. Alternatively,maternal chromosomes may be present but degenerate. Fluorescencemicroscopy of living human oocytes has shown that some oocytes which, atthe light microscope level, appear to have no chromosomes, when viewedusing electron microscopy, do in fact have a number of pyknoticchromosomes located throughout the cytoplasm [37].


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2.1.1.3 Parental Origin of PHM

PHM are distinct from CHM in that they are triploid [11]. Severalmechanisms may account for a triploid conceptus, the most likely beingfailure at the first or second meiotic division during gametogenesis to

produce a diploid sperm or ovum, or fertilisation of an ovum by two sperm.These mechanisms have all been shown to occur in triploid material fromspontaneous abortions [38-41]. Studies of cytogenetic and enzymepolymorphisms in molar tissue obtained clinically [14], or from a series ofspontaneous abortions [17] confirmed that PHM were triploid and in almostall cases diandric, the additional chromosome set being paternally derived.

The majority of these diandric PHM have been shown to arise by dispermy[14,17, 18], although cases have been described in which the most likelyorigin is fertilisation of an egg by a diploid sperm [42,unpublishedobservations]. Although PHM are diandric triploids, Zaragosa et al suggestedthat the converse may not be true and that not all paternally derived triploids

develop as PHM [42].

2.1.1.4 Genetically Unusual HM

Although most HM can be classified as CHM or PHM as described above,HM have been identified which are neither androgenetic diploids nordiandric triploids. Amongst CHM, rare tetraploid and triploid cases havebeen reported. However, they remain androgenetic in origin having 3 or 4paternal sets of chromosomes [43]. Tetraploid PHM have also been reported[18, 43-45]. Like triploid PHM, these HM generally have an excess of

paternal genomes. Their most likely origin is fertilisation of an egg by threesperm or two sperm, of which one is diploid. Two cases of tetraploid PHM

with apparently equal contributions from each parent have also beendescribed [18,46].

The morphology of the few aneuploid HM that have been reported[14,15,47,48] generally reflects their basic karyotype. Hypodiploid HM aremorphologically CHM while hypo- or hypertriploid HM are PHM. It hasbeen suggested that aneuploid populations may be present in a number ofCHM. Selective culture of stromal or trophoblast cells from CHM haveshown an aneuploid populations of cells in trophoblast cultures from mostCHM [49]. Similar observations have been made usingin situhybridisation to

pathological sections of CHM with chromosome-specific probes. Whilevillous trophoblast and stromal cells were found to be diploid, highproportions of polyploid cells were found in the extravillous trophoblast[50].

Occasionally areas of molar change may be seen in the placenta of anotherwise normal pregnancy. These rare cases which may comprise twodifferent cell lines that are both diploid [51], or a triploid cell line together

with a normal diploid line [52-54], are likely to represent unusual cases ofconfined placental mosaicism.


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2.1.1.5 Diploid, Biparental HM

A number of diploid PHM have been reported in the literature. Many ofthese may represent misdiagnosis of diploid hydropic abortion or CHM,particularly early CHM [55,56]. However, some cases of diploid PHM may in

fact fall into a rare, but extremely interesting subgroup of HM, whichalthough diploid have a normal or biparental genotype, with a geneticcontribution from both parents.

Diploid, biparental HM, first described by Jacobs et al [15], are usuallyclassified as CHM on the basis of histopathology [33,57-62], although onepatient with recurrent diploid, biparental HM of partial molar pathology hasbeen described [63,64]. More importantly, biparental CHM (BiCHM) arefrequently associated with patients who have repetitive CHM [61,62,65] andfamilies in which two or more individuals have molar pregnancies [61,65,66](2.1.1 below).

2.1.2 GESTATIONAL TROPHOBLASTIC TUMOURS

GTT have been less well studied than HM. Monitoring of disease bymeasurement of serum human chorionic gonadotropin (hCG) levels and thedevelopment of successful chemotherapeutic regimens has meant thatsurgical intervention, and hence fresh tumour tissue for investigation, is rare.

2.1.2.1 Invasive Mole

Cytogenetic studies of IM have shown the majority to be diploid with a highproportion of cells in the tetraploid range [5]. A detailed study of four IM by

Wake et al [30] found them to be diploid, three 46,XX and one 46,XYreflecting their origin from CHM. In one case the antecedent HM was alsoexamined and shown to be identical to the IM for all markers. All four IMappeared to have derived from dispermic CHM rather than the morecommon monospermic type. Two further cases of IM have been describedas aneuploid with a modal number of 46 [67].

2.1.2.2 Choriocarcinoma

Choriocarcinoma may follow any type of pregnancy; approximately equalnumbers following normal pregnancy, non-molar abortion or HM [68]. Thenature of the antecedent pregnancy will determine the chromosomalcomplement of the tumour. Tumours following term pregnancies, non-molar abortions or PHM will have both maternal and paternal chromosomes

while those deriving from CHM will be androgenetic in origin. Cytogeneticanalysis of choriocarcinoma cell lines and tumour tissue has shown a moreaberrant karyotype than that of IM with a wide variation in karyotype. Mostchoriocarcinoma are aneuploid with modes in the hyperdiploid and


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hypotetraploid range irrespective of whether they follow term births, HM ornon-molar abortions [5, 69-76]. Although karyotypes of choriocarcinomasshow a range of abnormalities, including chromosomal gains, losses andrearrangements, consistent chromosomal abnormalities have not been foundin cytogenetic studies. However, molecular genetic studies have

demonstrated frequent loss of specific regions of chromosome 7 and 8p[77,78], suggesting the presence of tumour suppressor genes in these regionsthat might play a role in the development of GTT. Similarly, amplificationof 7q [78] suggests a region of the genome where significant oncogenes maybe located. Further investigations are required to identify the specific genesinvolved.

2.1.2.3 Placental Site Trophoblastic Tumour

Very few data are currently available on the genetics of PSTT. Although

these tumours are less often associated with an antecedent molar pregnancythan choriocarcinoma, DNA analysis has shown that they may also arisefrom either HM or normal term pregnancy [79]. Cytogenetic analysis of asingle PSTT by Lathrop et al [80] showed it to be diploid.

2.2 GENOMIC IMPRINTING IN GESTATIONALTROPHOBLASTIC DISEASE

2.2.1 Hydatidiform Moles

A small number of genes are transcribed only from the maternally orpaternally inherited allele, the allele inherited from the other parent beingimprinted or silent. This phenomenon, genomic imprinting, underlies theabnormal pathology seen in HM. Several lines of evidence suggest that thecharacteristic pathological features shared by androgenetic CHM and PHMare due to the presence of two paternal genomes. Ovarian teratomas,genetically analogous to CHM, but with a maternally derived, diploidgenome [81] have a very different pathology to CHM, the development ofembryonic tissues being favoured in this condition. Similarly digynictriploids, which have two maternal contributions to the nuclear genome, arenot generally associated with molar pathology [17] but have an abnormally

small placenta and growth-retarded fetus [82]. Where only paternalchromosomes are present a CHM develops, trophoblastic hyperplasia ismarked and no fetus is present. In PHM the presence of a maternal genomeis associated with more moderate trophoblastic hyperplasia and fetaldevelopment. Thus both loss of maternally transcribed genes andoverexpression of paternally transcribed genes are likely to play a role inmolar development.

Further evidence for the association between the paternal genome andplacental development comes from mouse models [83,84]. Experiments thatinvolve nuclear transfer in mice have shown that normal development


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during mouse embryogenesis requires both maternal and paternalcontributions to the genome. In these models relatively good trophoblastdifferentiation occurs in androgenetic embryos with two male pronucleicompared with gynogenetic embryos having two female pronuclei [84]demonstrating an association between the paternal genome and

extraembryonic development analogous to that seen in CHM.Several imprinted genes have now been identified in man [85], and some

examined in molar tissue. Distribution of the maternally transcribed geneH19 in CHM tissue is controversial, but most studies conclude that H19 isexpressed in CHM despite their androgenetic origin [86-89]. Since H19 isfrequently biallelically expressed in early placental development [90] thesignificance of these observations is unclear at present. A second maternallytranscribed gene, CDKN1C, which shows high levels of expression innormal human placenta [91], is clearly repressed in the cytotrophoblast and

villous mesenchyme of CHM [92]. As a consequence, immunohistochemicalstaining with p57KIP2, the product ofCDKN1C, has proved to be a reliable

diagnostic discriminator between CHM and other types of pregnancies [93].The paternally transcribed gene, ZNF127, has also been examined in CHMand shown to exhibit the expected paternal methylation pattern [94].Conversely the androgenetic nature of CHM has made tissue from theseconceptions useful in the identification of novel paternally transcribed genessuch as ZAC/PLAGL1 and HYMAI[95,96].

2.2.1.1 Familial Hydatidiform Mole

Familial clustering of GTD is rare. The first report of familial HM described

3 unrelated Indian kindreds in which two or more individuals (sisters and acousin in the first, two and three sisters respectively in the second and third)presented with molar pregnancies [97]. Two Italian kindreds each with twoaffected sisters were subsequently described, one pair of sisters beingmonozygotic twins [98; 99]. Three further families have been reported in theliterature. The first, a Lebanese family in which 2 sisters and a cousin[63,100] were affected, the second, a German family in which three sistershad HM [101,102] and the most recent, two Italian sisters with HM [65].Consanguinity was noted in one family described by Ambani et al [97], theLebanese family [100] and the Italian family described by Sensi et al [65].Consanguinity was not reported in the remaining families although some

came from small rural communities. Most affected women had severalconsecutive HM with very few normal pregnancies being described in any ofthe affected women.

One patient from the Lebanese kindred, originally described by Vejerslevet al [63], had recurrent PHM. However, most familial HM are pathologicallyCHM [61,65]. More importantly all familial HM, in which genetic studieshave been done, have been shown to be diploid but biparental in originrather than androgenetic [61,65,66] suggesting that BiCHM might representa familial form of the condition. The pattern of inheritance in affectedfamilies suggests an autosomal recessive condition predisposing affected


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women to molar pregnancies. Since BiCHM are pathological lyindistinguishable from androgenetic CHM (AnCHM), in which theunderlying defect is abnormal genomic imprinting, the gene defect in these

women is also likely to result in deregulation of normal imprinting. Mappingof the gene defect in these families should lead to the identification of a

gene(s) important in the control of normal imprinting in both embryonicand extraembryonic development. A genome-wide scan of two familiesenabled Moglabey et al [66] to map the gene for familial HM to 19q13.3-q13.4 in a 15.2-cM interval flanked by D19S924 and D19S890, a region

which was subsequently narrowed by Sensi et al [65] to that flanked byD19S418 and D19S890. The specific gene that gives rise to familial HM hasyet to be identified and further studies are clearly needed to refine thelocation and identify the gene involved.

Although few cases of familial HM have been described, several isolatedcases of patients with recurrent HM have been reported, some patientshaving as many as 9 or 10 consecutive moles [103]. As in familial HM,

normal pregnancies are extremely rare in individuals with several recurrentHM. It has recently been demonstrated that the HM in these patients canalso be of biparental origin, [62] suggesting that these are individuals with thesame rare autosomal recessive predisposition to molar pregnancies.

2.2.2 Gestational Trophoblastic Tumours

The greatest risk factor for the development of GTT is a pregnancy withHM [68] in particular a CHM [104]. Despite this, the majority of CHM stillresolve spontaneously. One of the important questions in the management

of patients with HM is predicting those HM which will progress to GTT.Since histopathological grade has not been shown to have prognosticsignificance [105], the search continues for genetic factors, which mightidentify those CHM at risk. Since early studies of CHM showed them to behomozygous it was suggested that development of GTT might be related tohomozygosity for deleterious recessive genes in these conceptuses [19].Dispermic CHM that are homozygous for only some loci would be expectedto show less frequent progression to GTT. Since there is no evidence thatmonospermic CHM are more likely to progress to GTT, it may be thepaternal nature of the genome, rather than homozygosity for specific genes,

which is the important factor in post-mole tumourigenesis.

There is now both indirect and direct evidence for deregulation of normalgenomic imprinting in the development of some types of tumour.Preferential mutation of paternal alleles is associated with loss of tumoursuppressor genes in several childhood tumours [106,107], while anassociation between excess paternal genes and tumourigenesis is implicatedin Beckwith-Wiedemann Syndrome (BWS) [108]. More direct evidence of arole for imprinted genes in tumourigenesis comes from studies showing lossor gain of normal imprinting in tumour tissue. Following earlier reports ofloss of the parental imprint for both the maternally imprinted, IGF2and thepaternally imprinted, H19 genes in Wilms tumour [109,110], a number of


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tumours have been found to show loss, or relaxation, of imprinting at theseloci [111]. More recent studies have shown gain of imprinting (ie loss ofexpression) of these and other genes in meningiomas [112] andhepatocellular carcinoma [113]. Loss of maternally expressed genes or gainof paternally expressed genes could be related to the malignant potential of

CHM.Studies ofH19 and IGF2 expression in choriocarcinoma have shown

both to be expressed in post-mole choriocarcinoma [88,89,114], althoughthe level ofH19 in post-mole choriocarcinoma appeared to be less in thetumour tissue than the preceding CHM [88]. Studies ofH19 and IGF2 inchoriocarcinoma cell lines, some of which derive from tumours followingterm pregnancies, showed frequent biallelic expression of one or both genes[89,114]. Thus loss of imprinting may be an important factor in thedevelopment of GTT from molar and non-molar pregnancies. Studies ofgenomic imprinting in GTD should increase our understanding of the roleof imprinting in both embryonic and extraembryonic development and the

role it plays in the development of GTT.

2.3 GENETIC DIAGNOSIS

2.3.1 Differential Diagnosis in HM

2.3.1.1 Hydropic Abortion, PHM or CHM

It is estimated that the risk of choriocarcinoma following HM is in the orderof 1000 times more likely than after a normal conception [115]. Although it

has been confirmed that PHM can progress to choriocarcinoma [116],studies in which HM have been characterised as CHM or PHM have shownthat it is the patients with CHM that are likely to require treatment forpersistent trophoblastic disease (PTD) [9,10,18,104]. In the UKapproximately 1 in 12 patients with CHM require treatment for PTD [104],

which, in the absence of a pathological diagnosis, may be IM,choriocarcinoma or PSTT. Estimates of the percentage of patients withPHM which do not resolve, following evacuation of the molar pregnancy,range from 0.5% [117] to 5% [118]. However, even the lower of thesefigures is likely to be an overestimate due to the large number of PHM

which go undiagnosed [68, 55, 119]. Since the risk of developing PTD is

very different following CHM, PHM or hydropic abortion it is important todistinguish between these conceptions.

Although diagnosis is usually possible on the basis of morphology, poorsampling, or necrosis in a HM that has been retained for a long period, canmake a pathological diagnosis difficult [55]. The introduction of ultrasoundscanning and earlier termination of suspected molar pregnancies has alsopresented some diagnostic problems. At earlier gestational age thedistinction between CHM and PHM is less marked [55], with fetal tissuesuch as nucleated red blood cells, endothelial cells, stromal macrophages,amnion and yolk sac, characteristics once thought to be diagnostic of PHM,


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occasionally being present in early CHM [60, 120; 121]. Other conditionsthat may be pathologically difficult to distinguish from PHM, but whichmight be genetically diploid, include BWS [56,122] and placentalangiomatous malformation [56]. In such circumstances genetic studies canhelp in the differential diagnosis.

2.3.1.2 Twin Pregnancy with CHM and Coexistent Normal Fetus or PHM

Twin pregnancy consisting of CHM and normal fetus may also posediagnostic problems [123]. PHM are characterised by a range of villi fromnormal to cystic and show evidence of the presence of a fetus or fetaldevelopment, generally in the form of fetal red blood cells in the villi. Twinpregnancies consisting of normal twin and CHM may also show similarcharacteristics. It has been suggested that CHM in a twin pregnancy poses asignificantly greater risk of PTD than a singleton CHM and that the disease

tends to be more aggressive [124-126]. Although this has not beensubstantiated by a recent study of CHM with co-existing normal twin in

which 40% of women successfully delivered a normal baby withoutincreased risk of PTD [127], both the clinical outcome, which may be alivebirth, and the risk of PTD is likely to be very different in a twinpregnancy with CHM than a PHM. It is therefore important to make thedistinction between these two entities, both for appropriate management ofthe pregnancy and subsequent follow up. Several cases have been reported

where genetic studies have been used to identify CHM in twin pregnancies[128-136] or in multiple conceptions [137-142].

2.3.1.3 Monospermic, Dispermic or Biparental CHM

A distinction between monospermic and dispermic CHM can only be madeon the basis of genetic polymorphisms, there being no morphologicaldifference between these two conditions [143]. At present the clinicalsignificance of this distinction is not clear. Studies of choriocarcinoma tissueand cell lines cell lines have suggested that dispermic CHM have the moremalignant potential [30, 57,144-146]. However, when the incidence of PTDfollowing monospermic or dispermic CHM has been examined, nodifference in the relative incidence of PTD [18] has been identified. This is

supported by the observation that Y chromosome-positive CHM do nothave a greater chance of metastases than Y chromosome-negative CHM[147]. Larger studies are still needed to resolve this question.

As with monospermic and dispermic CHM, classification of CHM asbiparental or androgenetic can only be made by carrying out genetic studies.PTD has been reported following BiCHM [65,unpublished observations].However, the rarity of this condition makes it difficult to assess the relativerisk of PTD following BiCHM compared to AnCHM. Although rare,BiCHM pose an interesting diagnostic problem. Although most women whohave a HM go on to have normal pregnancies they are at an increased risk,


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approximately 1%, that their next pregnancy will be a HM [104,148-150].This risk increases greatly after two HM to approximately 20% [104,148].Women with recurrent HM may wish to consider IVF to avoid further HM.It has been suggested that for patients with recurrent HM, intracytoplasmicsperm injection (ICSI) followed by preimplantation genetic diagnosis can be

used to prevent molar pregnancies [151]. ICSI ensures only a single spermenters the egg, thus preventing dispermic CHM or PHM. Followingfertilisation, rejection of 46,XX embryos in favour of 46,XY embryos willeliminate monospermic CHM which arise by doubling of a haploid sperm[151]. However, this strategy will not be effective where the CHM are ofbiparental origin since these may be either male or female [62]. It is therefore

very important to distinguish between recurrent CHM of androgenetic orbiparental origin in patients considering IVF.

2.3.2 Diagnostic Problems in Trophoblastic Tumours

2.3.2.1 Gestational and Non-gestational Trophoblastic Tumours

GTT are a unique group of tumours in that they are allografts arising, notfrom the patient's own tissue, but from a genetically distinct conceptus. Theyare also unique in their response to cytotoxic drugs. GTT are characterisedmorphologically by the presence of cytotrophoblastand syncytiotrophoblastcells and biochemically by the production of hCG. A diagnosis of GTT maybe complicated because trophoblastic differentiation and hCG productioncan occasionally occur in non-gestational tumours [152]. Patients with non-gestational tumours showing trophoblastic differentiation may respond to

current chemotherapy but long term survival is uncommon.Using genetic techniques it is now possible to determine the gestational

or non- gestational origin of trophoblastic tumours. A tumour that isgestational in origin will reflect the genome of the pregnancy in which itarose. It will therefore have both paternal and maternal DNA if it derivesfrom a normal pregnancy or non-molar abortion, or paternal DNA if itoriginates in a CHM. In any case the presence of paternal genes willdistinguish it from a non-gestational carcinoma with trophoblasticmetaplasia which will have a genome which reflects that of the host. Geneticstudies have shown that metastatic choriocarcinoma in a variety of tissuesincluding, brain, lung and ovary [153-156 unpublished observations] may be

of non-gestational origin. Conversely a small number of tumours of theovary, which behaved clinically like primary choriocarcinoma, have beenshown to be gestational in origin [153, 157].

2.3.2.2 The Antecedent Pregnancy in GTT

The nature of the pregnancy in which a tumour arises cannot be determinedmorphologically but is important clinically since tumours that arise followinga HM have a more favorable prognosis than those which arise from a term


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pregnancy or non-molar abortion. The time interval between a pregnancyand the diagnosis of a tumour is also a factor in determining the appropriatechemotherapeutic regimen [158]. Therefore ascertainment of the nature ofthe causative pregnancy and the time interval between that pregnancy andthe diagnosis of the tumour will enable better patient management.

Clinically, the immediately antecedent pregnancy is perceived as thecausative pregnancy. However, in a patient with multiple pregnancies it isnot possible to be sure that the last recognised pregnancy is the one in whichthe tumour arose. A number of genetic studies have now shown that theantecedent pregnancy is not always the causative pregnancy in cases of GTT[146,153,154,159,160]. Patients may have tumours that are clearlyandrogenetic in origin following non-molar pregnancies, tumours that derivefrom a normal fertilisation after a molar pregnancy or tumours in which thesex chromosome complement is different to that of the antecedentpregnancy. Patients may in fact have more than one intervening pregnancybefore developing a GTD. We have identified one patient with three further

pregnancies in the eight year interval between her molar pregnancy and thesubsequent development of post-mole choriocarcinoma.

2.3.2 Diagnostic Techniques

2.3.2.1 Determination of Cell Ploidy

Ploidy of both HM and GTT has been determined by karyotype analysis.However, these techniques generally require cell culture and good qualitymetaphase spreads for analysis. An assessment of ploidy can be made by

other techniques. In particular, flow cytometry, a technique based on thelevel of fluorescence emitted from labeled nuclei, has been useful indistinguishing diploid CHM from triploid PHM [161-164] (Figure 2.1) andfor identifying the more unusual tetraploids [46]. One of the mainadvantages of flow cytometry is that it can be applied to cells recovered fromformalin-fixed, paraffin-embedded blocks [165]. Determination of cell ploidycan also be made by fluorochrome staining of interphase nuclei [166] or insitu hybridisation with chromosome-specific probes [50,167,168]. Triploidcells are distinguished from diploid cells by the presence of three spotsrather than two for each probe used.


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Figure 2.1 Flow cytometry of cells from (a) CHM and (b) PHM tissue. In the CHM amajor diploid (2n) peak is observed with a smaller tetraploid (4n) peak representing CHMcells undergoing division. In PHM a small diploid peak is present which corresponds tocontaminating host cells while the major triploid peak (3n) and smaller 6n peak representresting and dividing cells of the PHM respectively.

Flow cytometry and in situhybridisation have revealed more heterogeneity inCHM cells than previously found in most cytogenetic studies. Single casesof an apparently haploid CHM [18] and apparently haploid PHM [169] havebeen identified by flow cytometry. Some studies of cell ploidy have alsoidentified aneuploid or polyploid populations of cells in CHM[50,164,169,170]. It was suggested by Martin et al [170] that ploidycorrelated with clinical course. Although this was not confirmed bysubsequent studies [169, 164,171], a more recent study by Fukunaga et al[172] has suggested that aneuploid CHM are in fact associated with less riskof PTD than diploid or tetraploid CHM. Proliferative activity, higher inCHM than non-molar placenta, has not been found to be a useful predictorof prognosis [162,164,170,173].

Using flow cytometry, both choriocarcinoma [117,173-175] and PSTT[175-177] have generally been shown to be diploid despite the variedkaryotypes identified in choriocarcinomas by cytogenetic analysis. In somecases this may reflect the high proportion of infiltrating host cells present inpathological blocks of tumour tissue which will be diploid. Studies of cellploidy are very useful for establishing a differential diagnosis between CHMand PHM, or PHM and its pathological mimics, in problem cases [56,178].However, these techniques cannot distinguish between molar and non-molartriploids. Nor can they be generally be used in determining the origin orcausative pregnancy in GTT. Genetic studies of polymorphic markers arerequired to answer these questions.

2.3.2.2 Molecular Genetic Techniques

Major advances in the diagnosis and classification of GTD came with theintroduction of molecular genetic techniques that use DNA polymorphismsas a basis for determining the parental origin of molar or tumour tissue.

These polymorphisms, in particular the hypervariable minisatellite


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polymorphisms [179-181] were very much more informative than thecytogenetic or enzyme polymorphisms previously used [18,32]. Subsequentlyshorter variable DNA sequences, the microsatellite polymorphisms [182,183] were identified. Used in conjunction with the polymerase chain reaction(PCR), a technique for amplifying small amounts of DNA, these markers

have enabled rapid diagnosis from even small amounts of tissue. Thegreatest advantage of these techniques, particularly for the pathologist, wasthat the microsatellite sequences were short enough to be amplified even indegraded DNA prepared from pathological blocks [184-186]. Preparation ofDNA from pathological blocks also has the advantage that microdissectionof the cells of interest can be performed prior to DNA preparation thusminimising contamination of the trophoblastic tissue with DNA from hosttissue.

The most recent modification of these techniques has been theincorporation of a fluorescently labeled primer in the PCR reaction followedby automated sizing of the PCR products [185]. This allows a number of

polymorphisms from different regions of the genome to be examinedsimultaneously and provides a rapid means of determining genetic origin ofboth molar and tumour tissue. Further refinements have also come from theuse of laser capture microdissection to separate small areas of trophoblasticcells from surrounding host tissue in stained sections (Figure 2.2).

2.3.3.2.1 Molecular Genetic Diagnosis of Hydatidiform Moles

In early molecular genetic studies of trophoblastic disease, restrictionfragment length polymorphisms [59], DNA fingerprinting [31,187,188], and

the locus specific minisatellite probes [18,32,33,133, 189] were all used toconfirm the androgenetic origin of CHM, to distinguish monospermic fromdispermic CHM, to confirm the diandric origin of PHM and distinguishCHM with twin from PHM. Subsequently PCR amplification of sexchromosome-specific sequences [147,190] (Figure 2.3) minisatellite [135,190-192] and microsatellite polymorphisms [184,185] were also used in thedifferential diagnosis of HM (Figure 2.4). Molecular genetics has also proveduseful in distinguishing recurrent HM, ie a new conceptus, from PTD arisingin a previous HM [160,193].Studies involving comparison of microsatellite polymorphisms in parentaland molar tissue have been particularly important in increasing our

understanding of molar development, firstly in demonstrating the presenceof tissue of embryonic origin in early CHM [60,120,121] and secondly in theidentification of BiCHM [61,62,65].


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Figure 2.2 Laser-capture microdissection of trophoblast cells from formalin-fixed paraffin-embedded HM (a-c) and choriocarcinoma (d-f) tissue. (a) Unmounted, hematoxylin andeosin (H&E) stained section of CHM before microdissection. (b) Section following capture

of villous trophoblast. (c) Trophoblast cells retained on cap following laser-capture. (d)H&E stained section of needle core brain biopsy with focus of metastatic choriocarcinoma.(e) before and (f) after microdissection of trophoblastic cells from surrounding host tissue.DNA is prepared from the cells that are retained on the cap following microdissection.(courtesy of Samantha Thornton).

Studies involving comparison of microsatellite polymorphisms inparental and molar tissue have been particularly important in increasing ourunderstanding of molar development, firstly in demonstrating the presenceof tissue of embryonic origin in early CHM [60,120,121] and secondly in the

identification of BiCHM [61,62,65].

2.3.3.2.2 Molecular Genetic Diagnosis of GTT

Restriction fragment length polymorphisms of DNA [79,154,155,194-196],PCR amplification of minisatellite polymorphisms [159], sex chromosome-specific sequences [155,184] and microsatellite polymorphisms [146,184,197]have all been used to examine trophoblastic tumour tissue, to distinguish


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gestational from non-gestational tumours (Figure 2.5) and identify thecausative pregnancy in cases of GTT (Figure 2.6).

In the largest genetic study of trophoblastic tumours [146,155],approximately 10% of the cases were found to be non-gestational, whileapproximately half the remainder originated in CHM and half in HA or term

pregnancy. However, in at least 8 of the 33 cases of GTT the causativepregnancy was not the clinically antecedent pregnancy. In three theimmediately antecedent pregnancy was a CHM while the tumour resultedfrom a normal conception, despite the fact that the last normal pregnancy inone case was 16 years previously. In five cases, where the previouspregnancy was non-molar, two of the tumours had originated in CHM while3 were found to be of different sex to the antecedent pregnancy. Similarresults have been found in other studies (unpublished observations).

Although all post-mole tumours in the series described by Shahib et aloriginated in CHM [146], it has been formally demonstrated, using geneticstudies, that choriocarcinoma can arise in PHM [116].

Figure 2.3 PCR amplification of DNA with primers for sex chromosome-specificsequences [198]. DNA from the patient shows only the 130 bp X chromosome-specificband while DNA from her partner has both the X chromosome-specific and 170 bp Ychromosome-specific fragment. DNA samples prepared from a paraffin-embeddedpathological block of CHM and tissue from a subsequent choriocarcinoma are both Ychromosome-negative and therefore female.


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Figure 2.4 Typical, informative microsatellite polymorphisms identified in DNA from parental blood andmolar tissue following PCR amplification with fluorescently labeled primers. The sizes of the DNAfragments generated (in base pairs) are shown on the X axis. (a) A CHM, homozygous for the D13S317microsatellite marker, having a single allele that is the same size as one of the paternal alleles (shaded). Nomaternal DNA is present indicating that the HM is androgenetic. (b) A PHM, trisomic for the D20S481locus. The molar DNA has one allele from the patient and two alleles (shaded) from the father, consistent

with a diandric triploid conception. (c) A CHM which is disomic but in which one allele is inherited from thepatient (shaded) and one from the partner (shaded), consistent with a biparental origin.

Figure 2.5 Typical, informative microsatellite polymorphisms identified in DNA from parental blood andtrophoblastic tumour tissue following PCR amplification with fluorescently labeled primers. (a) A GTT withtwo alleles, one derived from each parent (shaded). (b) A non-gestational tumour with two alleles identical tothose in the patient (shaded) for the D21S1270 locus. For the D11S1999 locus, loss one of the two maternalalleles (arrowed) confirms that the tissue examined is tumour and not contaminating host cells.


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These results demonstrate the importance of genetic studies in themanagement of GTT. The ability to identify microsatellitepolymorphisms in fixed material now provides a rapid accuratetechnique that can be used to determine the origin of trophoblastictumours.

Figure 2.6 Fluorescently labeled products identified following amplification ofmicrosatellite markers in DNA from a patient, her partner, tumour and antecedentpregnancy. The pregnancy prior to the development of choriocarcinoma wasshown to have two alleles, one from each parent. The tumour was geneticallydifferent to the antecedent pregnancy having only a single paternally derived allele

(shaded) which was different to the paternal allele in the previous pregnancy. Thegenotype of the tumour was consistent with an origin in an androgeneticconceptus and was subsequently shown to be identical to that of the CHM in aprevious twin pregnancy with CHM and co-existent live born infant [127].

2.4 SUMMARY

Genetic diagnosis can be used to classify HM as PHM, monospermicAnCHM, dispermic AnCHM or BiCHM. There are manycircumstances where a genetic diagnosis provides confirmation of


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pathological findings or resolves diagnosis in pathologically unusualcases. One of the most recent applications of genetic diagnosis tomolar pregnancies is to differentiate between AnCHM and BiCHMin cases of recurrent CHM, a diagnosis that cannot be made on thebasis of pathology alone. Genetic diagnosis has also proved

important in the management of patients with trophoblastictumours, in particular the differential diagnosis between gestationaland non-gestational tumours, for which there is a very differentprognosis. The development of techniques based on PCRamplification of microsatellite DNA polymorphisms now enablesrapid, accurate diagnosis of both fresh and fixed tissue from GTD.HM represent an extreme example of genomic imprinting; BiCHM,in particular, provide a valuable resource for investigating the role ofgenomic imprinting in embryonic and extraembryonic development.

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