Genetic studies in hydatidiform mole with clinical correlations

Placenta (1987). 8, 77-88

Genetic Studies in Hydatidiform Clinical Correlations*

Mole with

SYLVIA D. LAWLER & ROSEMARY A. FISHER Institute of Cancer Research and the Royal Marsden Fulham Road, London SW3 63’3, UK

Hospttal,

Paper accepted 4.7.1986

INTRODUCTION Sex chromatin Cytogenetic interest in hydatidiform moles (HMs) began with the determination of their sex. Park (1957) was the first pathologist to make a systematic observation of the presence of sex chromatin in HMs. On the basis of this and other studies made between 1957 and 1969, it has been known for many years that, using sex chromatin as the criterion, the majority of HMs have two X chromosomes. Nevertheless, because sex chromatin was not found in every case, some cases could have been assumed to be of male sex (Lawler, 1984). Cytogenetics

The first description of chromosome studies in HM appeared in 1960 (Stolte et al, 1960); subsequently further studies were made in a number of populations. It was found that the majority of specimens diagnosed as HM or chorionic lesion, a term used by the Japanese workers to include HM, invasive mole and choriocarcinoma (Makino, Sasaki and Fukuschima, 1963), were 46,Xx, whereas the majority of HMs ascertained among spontaneous abortions were found to be triploid (review by Lawler, 1984). In 1977 Vassilakos, Riotton and Kajii divided HMs into two entities on the basis of morphological and cytogenetic studies. They studied a series of 81 I spontaneous abortions and 1097 induced abortions in which 75 specimens had gross swelling of the chorionic villi. Fifty-six of these specimens were classified as partial mole on the basis of pathology, showing normal chorionic villi interspersed with hydropic villi, the presence of a fetus, cord and/or amniotic membrane. Cytogenetically, 17 were classified as trisomic and 22 as triploid. The other 19 HMs were classified as complete moles on the basis of pathology and were without fetus, cord or amniotic membrane. The ten cases that could be karyotyped were 46,Xx. Further clarification of the categories of complete and partial mole was published by Szulman and Surti (r978a, r978b). Complete moles were defined as those having no fetus, total hydatidiform change from oedema to central cistern formation and conspicuous hyperplasia, while partial moles had a range of villi from normal to cystic, focal hyperplasia and evidence for the presence of a fetus. _ ??

Updated version of a paper presented at the Second World Congress on Trophoblastic Neoplasms, November 1984.

77

Placenta (1987). Vol. 8

78

They found seven cases of complete mole where sex chromosomes could be defined to be 46,Xx, and I I cases of partial mole showing triploidy. Androgenetic origin of complete hydatidiform moles In 1977 Kajii and Ohama, using chromosomal polymorphisms, established that the chromosomes in seven cases of 46,Xx complete mole were paternally derived. Thus, they demonstrated the androgenetic origin of complete moles. Kajii and Ohama suggested that the chromosome findings were ‘compatible with either fertilisation by a diploid sperm due to nondivision at second meiotic division or fertilisation by a haploid sperm followed by duplication of its chromosomes and nondivision at first “blastomere” mitosis’. Because a cell must have at least one X chromosome to survive, the predominance of XX complete mole was accounted for by non-viability of a YY conceptus. The androgenetic origin of complete mole was confirmed chromosomally by Wake, Takagi and Sasaki (1978) in three cases and by Jacobs et al (1978) in another three cases. Lawler et al (1979) used biochemical markers in the elucidation of the genetic origin of complete mole. The results confirmed the exclusively paternal origin of complete mole and were in favour of the origin being from duplication of a haploid sperm rather than a failure of meiosis II. Thus, up to 1979 complete moles had been found to be female, androgenetic and homozygous, but in that year Surti, Szulman and O’Brien (1979) described a paternally derived XY complete mole. Subsequently Ohama et al (1981) described the dispermic origin of 46,XY complete mole. Since then most heterozygous complete moles examined have been found to be 46,XY, but occasional examples of 46,Xx heterozygous complete mole have been described (Fisher and Lawler, 1984; Wake et al, 1984). Mitochondrial DNA of complete mole The mitochondrial deoxyribonucleic acid (DNA) of complete mole has been shown to be maternally derived (Wallace et al, 1982; Edwards et al, 1984) as it is in the normal conceptus (Giles et al, 1980). Thus, the complete mole represents a unique human cell type, combining a paternal nuclear genome with a maternal mitochondrial genome.

MATERIALS

AND METHODS

A follow-up study of patients after pregnancy with hydatidiform mole of defined type Fresh tissue for cytogenetic, biochemical marker and DNA studies was collected from patients undergoing termination of pregnancy because a diagnosis of HM was suspected on clinical grounds. All cases in which the diagnosis of HM was confirmed by pathology have been included in the series. Blood samples were obtained from the patient and her spouse. Methods of collection, storage and culturing of material for pathological, cytogenetic and biochemical marker studies have been recorded previously (Lawler et al, r982a, r982b). Hydatidiform moles in our series were classified as partial hydatidiform mole (PHM) or complete hydatidiform mole (CHM) on the basis of the pathological criteria described by Szulman and Surti, r978a, r978b. Cytogenetic analysis and/or flow cytometry or DA/DAPI staining (Tommerup and Vejerslev, 1985) have been used to determine the ploidy of cells from the molar tissue, while chromosome banding patterns, biochemical markers and more recently restriction fragment length polymorphisms have been used to determine the genetic origin. Up until the end of 1985, among 163 cases of hydatidiform mole, 38 patients have been classified as having PHM and 125 as having CHM.

Lnwlrr, Fisher: Genetic Studier in Hydatidi/rm Mole

RESULTS Partial moles (PHM) Table I shows the results of studying biochemical markers in a typical PHM. The informative loci were phosphoglucomutase-r (PGM,), acid phosphatase-r (ACP,), glyoxylase (GLO), phosphoglucomutase-3 (PGM,) and phosphoglycolate phosphatase (PGP). The molar tissue was found to be heterozygous at five loci, a trisomic state was demonstrated at four loci, and both maternal and paternal contributions were present at the ACP, locus. Thus the biochemical studies showed triploidy and the presence of maternal and paternal genes. Cytogenetic polymorphisms are generally more informative than biochemical markers in determining the origin of PHM. One of the cases of PHM in which the origin could be deduced is illustrated in Figure I. The number I chromosome of the patient, the PHM and the husband were stained to show centromeric polymorphisms so that all the paternal and maternal chromosomes were distinguishable. The mole was triploid 69,XXY. In deducing the origin of the PHM, the presence of an X and a Y chromosome excludes error at the second paternal meiotic division. The presence of one maternal and two similar paternal number I chromosomes Table I. Biochemical polymorphisms in PHM (case 15) PGM,

ACP,

GLO

PGM,

Patient

z-t

LMole

2-2-I Z-I

2 2-2-t 2-t

2-1

spouse

CB CBA BA

PGP

I 2-t-t 2-t

PGM, = phosphoglucomutase-t; ACP, = acid phosphatase-1; GLO = glyoxylase; PGM, = phosphoglucomutase-3; PGP = phosphoglycolate phosphatase.

1

Father 46tXY

lull PI

p2

PI

Pl

Mole 69,XXY

f‘l n

Mother 46,Xx

kigure I. Lateral asymmetry of chromosome I in PHM (case 17).

Placenta (1987). Vol. 8

80

excludes error at maternal meiosis and at the first paternal meiotic division. Therefore, the conceptus arose from the fertilization of a haploid egg by two sperms. The findings in the 38 cases of PHM in this series are summarized in Table 2. Twenty-three were shown to be regular triploids by karyotypic analysis, while two were found to be triploid when examined by flow cytometry. Two cases were hypertriploid, one being 7 I ,XXY, + C, + C and the other 7o,XXY, + mar. Where the sex of the mole was ascertained IZ were found to be XXY, I I XXX and one XYY. In our series the majority of PHMs (16/23) were proved to have arisen by dispermy. In the remaining seven dispermy was possible in all cases, although in two cases an origin by failure at the first paternal meiotic division was statistically more likely and in a further two origin by failure at the second paternal meiotic division was probable. In two cases an origin by a failure at the first paternal meiotic division was possible and in one an origin by digyny could not be excluded. Complete mole (CHM) Table 3 shows the results of studying biochemical markers in a typical CHM. The informative loci were PGM,, ACP,, GLO and esterase D (ESD). The CHM was shown to be homozygous at four loci where the male parent was heterozygous. A maternal contribution to the genome was excluded at the PGM, locus. Figure z shows the Q-band polymorphisms in a typical CHM. In this case a maternal contribution to the genome was excluded for chromosome numbers 3, 13, 15,21 and 22. The mole was shown to be homozygous for the polymorphisms of five autosomal pairs for which the father was heterozygous and the sex chromosomes. The findings in the 125 cases of CHM in this series are summarized in Table 4. Most of the CHMs (73/81) tested for genetic markers were shown to be homozygous for one or more genetic markers for which the father was heterozygous. Eight of the 81 (IO per cent) were found to be heterozygous for one or more genetic markers. Of the eight heterozygous CHMs five were 46,XY, with origin by dispermy proven in two previously published cases (Fisher, Sheppard and Lawler, 1984) and two new cases, while one was 46,Xx and shown to have arisen by Table 2. Partial hydatidiform mole: summary of 38 cases’ Pathological diagnosis of partial mole found to be triploid found to be hypettriploid not examined

38 25

Genetic origin examined origin by dispermy possible origin by dispetmy proved

23 23 16

2

II

’ Up to 31 December 1985

Table 3. Biochemical polymorphisms in CHM (case 24) PGM,

ACP,

GLO

ESD

I

BA A BA

*--I

I

Patient Mole

7

Spouse

7-1

PGM, = phosphoglucomutase-t; = glyoxylase; ESD = esterase D.

ACP, = acid

2 *-_I

phosphatase-t;

I 2-I

GLO

Latvkr, Fisher: Genetic Studies II Hydatidiforn

81

Mole

Fjgure z. C&band polymorphisms in homozygous CHM (case 101).

Table 4.

Completehydatidiform mole: summary of 125 cases’

Informative when tested for genetic markers

81

Homozygous moles --with exclusion of maternal contribution

73 47

Heterozygous moles --with exclusion of maternal contribution

8 6

a Up to 3’ December 1985.

dispermy (Fisher and Lawler, 1984). The two cases not karyotyped were heterozygous for biochemical markers. One was Y-body positive and the other Y-body negative. Figure 3 shows the Q-band polymorphisms in the female heterozygous CHM. Maternal exclusion was demonstrated in most chromosome pairs. The molar tissue was shown to be homozygous for the polymorphisms of four chromosome pairs (i.e., 3, 15, zz, X) and heterozygous for four chromosome pairs (i.e., 13, 14, 21 and 9) for which the father was heterozygous. Occasionally CHM can present with a fetus. This can happen if in a twin pregnancy one of the twins develops normally and the other forms a complete mole (review by Fisher, Sheppard and Lawler, 1982). Genetic studies can be used to determine the origin of such twins. We have ascertained two cases of mole with twin in our study. In one, the pregnancy was terminated for suspected hydatidiform mole (Fisher, Sheppard and Lawler, 1982). In the other the fetus was live-born, and the presence of CHM was not detected until delivery. In both cases the fetus was a normal 46,XY male. Recently Ohama et al (1985) have reported two more cases of molar placenta with a fetus which have been analysed using cytogenetic and enzyme polymorphisms and found to be dizygotic twins with an androgenetic mole and normal conception.

Placenta (1987)~ Vol. 8

Figure 3. Qband

polymorphisms

in heterozygous

CHM (case92)

Comparison of clinical findings in PHM and CHM Figure 4 shows the age distribution of 137 of the patients with HM in the study. It is known that the chance of a molar pregnancy increases with age (Stone and Bagshawe, 1979). In this series, the older patients were more likely to present with a CHM, all four patients over the age of 45 having CHM. Figure 5 shows the menstrual age of 129 of the molar pregnancies and demonstrates that the longest gestation times were associated with PHM. This is partly related to the fact that diagnosis is complicated by the presence of a fetus. lo-

-

Complete

111Partial

8-

18

22

Figure 4. Age distribution

26

30 34 38 Age (years)

moles

moles

42

of patients with molar pregnancies

46

n = 106

n =31

50

54

Lawler. Fisher: Genetic Studier in Hydatidiform Mole -

Complete Partial

moles moles

n = 100 n = 29

10 5 2 g ;;:

8642O6

8

10

12

14 16 18 20 22 Menstrual age (weeks)

Figure 5. Distribution

of menstrual

24

26

28

30

32

age in molar pregnancies

Comparison of patients requiring or not requiring chemotherapy after evacuation of mole

After the evacuation of the HM the patients were followed up, to detect persistence of trophoblastic activity, by monitoring the levels of human chorionic gonadotrophin (hCG). The requirement for intervention with chemotherapy was based on the criteria of Bagshawe et al (‘973). It is shown in Table 5 that in our series resolution of the trophoblastic disease subsequent to evacuation was spontaneous in all 38 patients who had PHM. On the other hand, 17.6 per cent of the patients with CHM subsequently required chemotherapy. This may be for invasive mole or choriocarcinoma. Since the requirement for treatment is based on the hCG levels of the patient, a pathological diagnosis of the residual trophoblastic disease is not always possible. In Table 6 the patients with CHM have been divided according to whether the molar pregnancy was a first or subsequent one. It can be seen that where a CHM is the patient’s first

Table 5. Outcome of hydatidiform

Patient received chemotherapy

Spontaneous resolution Complete Partial Total a Up to 31 December

Table 6. Obstetric

22

‘03 38 141

Total ‘a5 38 ‘63

0 22

1985.

history of 106 patients with complete mole Previous pregnancies

Spontaneous resolution Treatment required

mole”

53 9

Primignvidae 34 IO

Total 87 19

Placenta (1987). Vol. 8

84

pregnancy she is more likely to require treatment

for post-mole trophoblastic

disease (22.7 per

cent) than if she has had other pregnancies (14.5 per cent).

The data on patients’ ages illustrated in Figure 6 suggest that in this series the chance of a patient with a CHM requiring treatment increases above the age of 35. On the other hand there was no obvious effect of menstrual age in relation to resolution of molar pregnancy in patients with CHM (Figure 7). A comparison of the generic findings in relation to the outcome of a pregnancy with CHM is given in Table 7. Twenty-two (17.6 per cent) of patients with CHM have required treatment. Of these, 15 CHMs were tested for genetic markers. Thirteen were found to be homozygous and two heterozygous. A similar ratio of homozygous to heterozygous CHM, 6o:6, was found in patients not requiring further treatment. Thus in our study there is no obvious difference in the genetic origin of those CHMs in patients that require subsequent treatment and those in whom the molar pregnancy resolves spontaneously.

lo-

Total n = 106 for post-mole m Treatment trophoblastic disease n = 19

8C 6& $ E

4-

rl

2-

0

-Ir;l 14

,I,

18

22

26

30 34 38 Age (years)

42

46

50

54

Figure 6. Age distribution of patients with CHM.

14-

-

Total n = 100

12-

a

Treatment for post-mole trophoblastic disease n = 18

lob 8z $ &

64r

.s 8

10

12

14

16

18

20

22

24

Menstrual age (weeks) Figure 7. Distribution of menstrual age in CHM

26

28

30

32

Lawler. Fhcr:

Genetic Studws in Hydatidifm

Molt

85

Table 7. Summary of genetic studies in 125 cases of complete mole’

Number of cases with no further treatment after evacuation

Number of cases having chemotherapy after evacuation

103

22

66 6.2 6

‘5 ‘3 2

Total Informative genetic markers Homozygous CHM Heterozygous CHM ’ Up to 31 December 1985.

DISCUSSION In this study partial Twenty-two

hydatidiform

moles (PHMs)

have been shown to be in the triploid

out of 23 cases where the genetic origin was examined

maternal

and two sets of paternal

possible. majority

Thus triploid partial moles were characterized by an excess of paternal genome, the having arisen by dispermy, a minority by failure at the first or second paternal meiotic

division. Tetraploids

chromosomes;

range.

have been proved to have one

with an excess of paternal

chromosomes

al, 1982; Surti et al, 1986); in the two recently as PHM. In our experience disease.

However,

no patient

1976; Szulman

described

diagnosis and Surti,

contributions

have also been described cases these were classified

with PHM has yet required

our series is still relatively

patient having a pathological Bagshawe,

in one case two maternal

small. There

treatment

(Sheppard

1982; Mostoufi-Zadeh

for residual

required

trophoblastic

treatment

and Driscoll,

et

pathologically

have been cases described

of PHM has subsequently

were

where a

(Stone and

1985), although

it

should be noted that no unequivocal cases of choriocarcinoma following PHM have been reported (Szulman and Surti, 1985). When considering the relative requirement for treatment following termination of molar pregnancy it is of obvious importance to distinguish between PHM

and complete

Like PHM,

hydatidiform

the CHMs

mole (CHM)

with a normal

twin conceptus.

in our series were found to have two paternal

sets of chromosomes.

Unlike PHM, characterized

CHMs are diploid and have no maternal chromosomes. Thus complete moles are by a purely androgenetic constitution. Most CHMs were found to be

homozygous,

arising

by fertilization

of an empty

egg by a duplicated

CHMs (IO per cent) in our series were found to be heterozygous, examined these were found to arise by dispermy. Similar incidences other series (Davis et al, 1984; Wake et al, 1984). In our series patients

13/73 (17.8 per cent) of patients

with heterozygous

mole trophoblastic

tumour.

patients with homozygous requirement for treatment

with homozygous

mole (25 per cent) have required Thus

there was no statistically

haploid

sperm.

Eight

and where the origin was have recently been cited in CHM

subsequent significant

and two of the eight treatment

difference

for post-

between

the

CHM and those with heterozygous CHM in our series as far as the was concerned. However, the number of cases studied by us is small,

and there is evidence that heterozygous CHM might in fact have a more malignant potential. A high incidence of heterozygosity has been found in trophoblastic tumours that follow molar pregnancies. This has been shown in invasive mole (Wake et al, 1984) and in a choriocarcinoma and cell lines in which the antecedent pregnancy was with CHM (Wake et al, 1981; Sasaki et al, 1982; Sheppard, Fisher and Lawler, 1985).

Placcnra (1987), Vol. 8

86

Twenty-four patients with heterozygous moles with adequate follow-up, including the cases in the present series, have now been described (Kajii, 1980; Ohama et al, 1981; Surti, Szulman and O’Brien, 1982; Davis et al, 1984; Fisher, Sheppard and Lawler, 1984; Kajii et al, 1984; Wake et al, 1984). Nineteen were 46,XY, eleven were diagnosed as male on the basis of the presence of a Y body, two cases were 46,Xx, one case was Y-body negative, and in one case sex was not determined. Eleven of these patients (32 per cent) have subsequently required treatment with chemotherapy. It is possible that the requirement for treatment in cases of heterozygous moles is higher in the Japanese population because Wake et al (1984) found 60 per cent of their patients with heterozygous CHM were treated compared to 25 per cent in our current UK series. Another point of interest is the lower frequency of heterozygous female than male CHM that have been diagnosed. It could be that some cases of XX, heterozygous mole are not diagnosed because insufficient polymorphisms are informative. A CHM should not be considered to be proven homozygous unless five chromosomes for which the male parent is heterozygous are homozygous in the molar tissue. More studies are required, in different populations, of the genetics of CHM and follow-up of the patients after evacuation of mole. When a patient has a molar pregnancy it is important to ascertain not only whether the mole should be classified as partial or complete but CHM should be divided into heterozygous and homozygous types.

SUMMARY In an elective study of 163 hydatidiform moles 38 were classified as partial mole (PHM) and 125 as complete mole (CHM) on the basis of pathology. Genetic studies showed the PHM to be triploid with one maternal and two paternal chromosome sets. In all cases of PHM the molar pregnancy resolved spontaneously after evacuation. On the basis of genetic studies CHM which were diploid could be subdivided into two entities: homozygous androgenetic CHMs that were 46,Xx, and heterozygous CHMs which were androgenetic and usually 46,XY. In informative cases in this series the frequency of heterozygous CHM was IO per cent. Twenty-two (17.6 per cent) of all the patients with CHM required subsequent chemotherapy for post-mole trophoblastic tumour. Where patients with CHM could be classified as having homozygous or heterozygous CHM the requirement for treatment (17.8 per cent and 25 per cent, respectively) was not found to be significantly different in the two groups.

ACKNOWLEDGEMENTS We would like to thank the many consultants who have kindly sent us tissue from molar pregnancies. We are also grateful for the help of Mr D. M. Sheppard in examining the chromosome polymorphisms, Dr S. Povey and Mr S. Jeremiah in examining the enzyme polymorphisms, and Dr A. Szulman for advice on the pathology. We thank Professor K. D. Ragshawe and Miss Joan Dent for details of clinical follow-up and Mrs Maria De Mourn for help in preparation of the manuscript. This work was supported by a Medical Research Council Grant to the Fetal Tissue Bank and the joint Medial Research Council/Cancer Research Campaign funding of the Section of Human Genetics, Institute of Cancer Research.

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Lawltr, Fisher: Genetic Studies in Hydaridsform Molt

87

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Wake, N., Seki, T., Fujita, H. et al (1984) Malignant potential of homozygous and heterozygous complete moles. Cancer Research, 4, 122br230. Wallace, D. C., Surti, U., Adams, C. W. & Szulman, A. E. (1982) Complete moles have paternal chromosomes but maternal mitochondrial DNA. Human Genetics, 61, 145-147.