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Japanese Journal of Clinical Oncology Pages 504-508

Gestational Choriocarcinoma Whose Responsible Pregnancy was a Complete Hydatidiform Mole Identified by PCR Analysis with New Sequence Tagged Site Primers
Introduction
Case Report
   Clinical History
   Histopathological Findings
   Determination of the Pregnancy Responsible for the Choriocarcinoma
Discussion
References
Gestational Choriocarcinoma Whose Responsible Pregnancy was a Complete Hydatidiform Mole Identified by PCR Analysis with New Sequence Tagged Site Primers

Gestational Choriocarcinoma Whose Responsible Pregnancy was a Complete Hydatidiform Mole Identified by PCR Analysis with New Sequence Tagged Site Primers

Yoshibumi Kaneta1, Reiko Yoshiyama1, Noboru Inagaki1, Kiwamu Toyoshima1, Kimihiko Ito1, Ruriko Nishino1, Hirokatsu Kitai1, Hidenori Kato2, Kazuo Asanoma2 and Norio Wake2

1Department of Obstetrics and Gynecology, Social Insurance, Saitama Hospital, Urawa and 2Department of Obstetrics and Gynecology, Medical Institute of Bioregulation, Kyusyu University, Beppu, Oita, Japan

We report a case where the pregnancy responsible for a gestational choriocarcinoma was not the antecedent pregnancy or the second normal term delivery, but a complete hydatidiform mole that had advanced to clinically invasive mole. This responsible pregnancy was identified by polymerase chain reaction analysis (PCR). PCR analysis was performed by using five new sets of sequence-tagged site (STS) primers on four chromosomes (chr. 1, D1S225; chr. 3, D3S1744; chr. 12, D12S1090; chr. 18, D18S849 and D18S877). The constitution of alleles of choriocarcinoma was shown to be almost identical with that of the husband on every marker. The allele patterns of choriocarcinoma on D3S1744 and D12S1090 were not observed with DNA from the patient. The band pattern originating from molar DNA was also identical with those of the husband and choriocarcinomas on D18S849 and D1S225.

Key words: choriocarcinoma - polymerase chain reaction (PCR) - sequence tagged site (STS) primer

INTRODUCTION

Recently, hydatidiform mole and invasive mole have been well managed in Japan and the occurrence of postmolar choriocarcinoma has been decreasing (1). Up to now, the pregnancy responsible for choriocarcinoma has been thought to be the antecedent pregnancy or a hydatidiform mole. Now, thanks to advances in DNA analysis, the pregnancy responsible is being identified and a series of cases have been reported (2-11). The origin of choriocarcinoma has been evaluated by the restriction fragment length polymorphism method, variable-number tandem repeat method, sequence-specific oligonucleotides method and single-strand conformation polymorphism method.

In this paper, we report a case of gestational choriocarcinoma in which the responsible pregnancy was not the antecedent pregnancy or normal term delivery, but clinically invasive mole (12) originating from a hydatidiform mole as identified by PCR analysis using five new STS primers: D1S225, D3S1744, D12S1090, D18S849 and D18S877.

CASE REPORT

Clinical History

The first pregnancy ended in spontaneous abortion at 21 years old. The second resulted in a live birth, male, at 23 years old. The third pregnancy was diagnosed as spontaneous abortion and dilatation and curettage (D&C) was performed at 25 years old. H-E-stained sections of the intrauterine contents demonstrated complete hydatidiform mole (Fig. 1). After the operation, detailed examination led to a diagnosis of clinically invasive mole based on the criteria of the Japan Society of Obstetrics and Gynecology and the Japanese Pathological Society (12). Six courses of etoposide were given and the patient showed a complete response. The fourth pregnancy resulted in a live birth, male, at 29 years old.


Figure 1. Microscopic appearance of the complete hydatidiform mole. H-E, ×100. The tumor consisted of hydropic chorionic villi. There are no vessels in the stroma.

The second D&C was performed under the diagnosis of incomplete abortion at 33 years old and H-E-stained sections of the intrauterine contents showed choriocarcinoma (data not shown). Pelvic magnetic resonance imaging (MRI) showed an abnormal mass (Fig. 2). Computed tomography (CT) of the chest showed multiple metastases (data not shown). The cytopathological diagnosis in the cavity of the uterine corpus was class V, choriocarcinoma (data not shown). The clinical stage of the choriocarcinoma was stage IIIC (FIGO, 1991) (13). The patient was treated with primary therapeutic agents, three courses of MEA (methotrexate, etoposide, actinomycin D) (Table 1A). After chemotherapy, total abdominal hysterectomy and bilateral salpingo-oophorectomy were performed. The patient was treated with one additional course of MEA, but the serum [beta]-hCG-CTP level did not decrease (normal <0.7 mIU/ml) (Fig. 3) and the patient was treated with secondary chemotherapeutic agents, MAC (methotrexate, actinomycin D, cyclophosphamide) (Table 1B).


Figure 2. MRI of the pelvis revealed an abnormal mass, 6 cm in diameter, in the anterior wall of the uterine corpus that showed a high signal pattern outside and an irregular low signal pattern inside on the T2-weighted images.


Figure 3. Clinical course and the changes in urinary hCG, serum [beta]-hCG-CTP and serum SP-I.

Table 1. Chemotherapy regimen
(A) MEA regimen
Day 1: Methotrexate 150 mg/body (bolus, IV) and 300 mg/body DIV
Actinomycin D 0.5 mg/body (bolus, IV)
Etoposide 100 mg/body, DIV
Days 2-5: Actinomycin D 0.5 mg/body (bolus, IV)
Etoposide 100 mg/body, DIV
Leucovorin rescue:
Day 2: 15 mg (IM) twice/day  
Day 3: 15 mg (IM) once/day  
(B) MAC regimen
Days 1 - 4: Methotrexate 0.4 mg/kg/day (IM)

Actinomycin D 10 µg/kg/day (IV)
Cyclophosphamide 2-4 mg/day (IV)
Abbreviations: IV, intravenous injection; DIV, drip intravenous injection; IM, intramuscular injection.

Histopathological Findings

The uterus weighed 270 g and contained a tumor 5 cm in diameter in the anterior wall. It was necrotic in the center and there were viable cells in the periphery (data not shown).

H-E-stained sections of the uterus demonstrated massive coagulation necrosis and choriocarcinoma (Fig. 4). Testing for immunohistochemical markers showed that the tumor cells were positive for hCG (Fig. 5).


Figure 4. Microscopic appearance of the choriocarcinoma. H-E, ×100. The tumor showed malignant cells similar to cytotrophoblastic cells; some showed mitosis with coagulative clots and decidua.

Figure 5. Microscopic appearance of the choriocarcinoma immunohistochemically stained for hCG. ×100.

Determination of the Pregnancy Responsible for the Choriocarcinoma

With the consent of the patient and her husband, DNAs from paraffin-embedded blocks of tumor and peripheral blood leukocytes of the patient, her husband, the first child (male) and the second child (male) were obtained. The DNAs were extracted by incubation for 12 h with 0.5% sodium dodecyl sulfate (SDS) and 50 µg/ml proteinase k following phenol-chloroform extraction. Five sets of STS primers on four chromosomes (D1S225, F, 5[prime]-TGGCCTGAATAGACCATAAA A-3[prime]; R, 5[prime]-GCCTGGGTGACAAAGCA-3[prime]; D18S849, F, 5[prime]-AGGTCCCAGCCATATAGAGG-3[prime]; R, 5[prime]-GCCCTAGAACCAGGGATTAA-3[prime]; D18S877, F, 5[prime]- GATGATAGAGATGGCACATGA-3[prime]; R, 5[prime]-TCTTCATACATGCTTTATCATGC-3[prime]) from the Whitehead Institute (1997) and D3S1744 and D12S1090 as Quick-typeTM from Lifecodes Inc., were chosen for the analysis. For PCR amplification, the forward primer was end-labeled with [[gamma]-32P]dATP by T4 DNA polynucleotide kinase (Boehringer Mannheim, Germany). The PCR reactions were carried out using 100 ng of genomic DNA, 1.5 pmol of [gamma]-32P-labeled forward primer and the same amount of unlabeled reverse primer by 33 cycles of 94°C for 60 s, 53°C for 45 s and 72°C for 90 s. After the PCR reactions, the samples were electrophoresed on 6% denaturing polyacrylamide gels and the radioactivity was measured using a BAS 1000 analyzer (Fujix, Osaka, Japan).

Because the efficiency of recovery of DNA from the paraffin-embedded complete hydatidiform mole tissue was fairly low, only two markers could be analyzed on molar DNA.

The constitution of the alleles of choriocarcinoma was shown to be almost identical with that of the husband on every marker. The husband's (Hd) and choriocarcinoma's (Cho) patterns are bc/de and bb[prime]/ade on D1S225. On D3S1744, D18S849 and D18S877 both (Hd and Cho) of the banding patterns were identical (a/b, b/c and a/c, respectively). The allele patterns of choriocarcinoma on D3S1744 and D12S1090 were not observed with DNA from the patient. The band pattern originating from molar DNA was also identical with that of the husband and choriocarcinoma: b/c on D18S849 and bc/d on D1S225 (Table 2, Fig. 6).

   A
   C
   B
   D
   E

Figure 6. PCR patterns detected by hybridization from the patient (Pt), the husband (Hd), the first child (C1), the second child (C2), complete mole tissue (M) and choriocarcinoma tissue (Cho) with D1S225. A, D1S225 locus. Six informative bands are labeled a through e and b[prime], as shown on right of the lanes. B, D3S1744 locus. Three informative bands are labeled a through c. C, D12S1090 locus. Four informative bands are labeled a through c and a[prime]. D, D18S849 locus. Three informative bands are labeled a through c. E, D18S877 locus. Three informative bands are labeled a through c.

Table 2. PCR analysis of complete mole, choriocarcinoma, parents and some of their children
  D1S225 D3S1744 D12S1090 D18S849 D18S877
Pt bc/a c/c c/c a/c b/c
Hd bc/de a/b a/b b/c a/c
C1 c/de a/c b/c b/c ND
C2 bc/a b/c b/c a/b a/c
M bc/d ND ND b/c ND
Cho bb[prime]/ade a/b a/a[prime] b/c a/c
Abbreviations: Pt, patient; Hd, husband; C1, first child; C2, second child; M, complete mole tissue; Cho, choriocarcinoma tissue; ND, not determined.

DISCUSSION

The pregnancy responsible for choriocarcinoma was previously thought to be the antecedent pregnancy or a hydatidiform mole. Recently progress in DNA analysis has permitted the identification of the pregnancy responsible for gestational and non-gestational choriocarcinoma. This has allowed determination of the latent period of choriocarcinoma and has permitted a choice of treatment and prediction of the clinical course.

Previous studies (2-11) utilized various DNA analysis methods. Our case is the 11th report to identify the pregnancy responsible for choriocarcinoma by DNA analysis. However, our case is the first report of the use of new STS primers, D1S225, D3S1744, D12S1090, D18S849 and D18S877. These primers were employed because they can differentiate the origin of alleles because of the high heterogeneity (80-95%) and by using the PCR method we can analyze the genetic origin with a small amount of DNA, especially from paraffin-embedded samples.

Arima et al. (9) had classified the genetic origin of choriocarcinomas into three categories based on the results of DNA polymorphism analysis. The first category of origin was from androgenesis (homozygous mole and heterozygous mole). The second origin was from live births and abortion. In this category, a choriocarcinoma had alleles contributed by both parents. The third category was non-gestational choriocarcinoma, which lacked a paternal contribution, suggesting a parthenogenetic origin. Our case belonged to the first category.

Before the PCR analysis in this case, we could not identify which was the pregnancy responsible for the choriocarcinoma development, but the analysis of the D1S225 and D18S849 loci by PCR with STS primers suggested that the responsible pregnancy was not the second normal term delivery or antecedent pregnancy, but the complete hydatidiform mole that had advanced to clinically invasive mole. Although we could not obtain the amplified bands from the complete hydatidiform mole DNA on D3S1744, D12S1090 and D18S877 loci, identical banding patterns on D1S225 and D18S849 with the choriocarcinoma, and especially a lack of identity between these tumors and the patient, may support the above conclusion.

On D1S225, band e is a two base-pair smaller band (one repeat smaller because this sequence-tagged site primer consists of a dinucleotide repeat) associated with the appearance of band d, and we concluded that they came from the same chromosome allele. Band b[prime] was a one or two repeat larger band only observed in the choriocarcinoma. It is likely that the band derived from the husband's allele and was generated by the genetic instability of frequently observed cancers. On D12S1090, one band a in the choriocarcinoma was identical with the husband's, but the other was different from any bands observed in either the husband or the patient. It was possibly derived from the instability as observed on D1S225.

The antecedent pregnancy had been tentatively determined as the origin of a choriocarcinoma. However, our PCR analysis for determining the genetic origin demonstrated that not the antecedent but the former molar pregnancy was the origin of the choriocarcinoma. Recently, the technology of molecular biology has advanced substantially and the techniques involved have become more popular and easier. The correct genetic origin of choriocarcinoma should be determined by analyzing the allele constitution using techniques such as molecular biological methodology. The accumulation of these data will provide new material for the classification, treatment and management of choriocarcinomas.

References

1. Hara T, Kaseki S, Tomoda Y, Nishikawa Y, Ishizuka T, Ishizuka N. Determinants of risk for developing invasive mole and choriocarcinoma following hydatidiform mole. Asia-Oceania J Obstet Gynecol 1986;12:241-50.

2. Fisher RA, Lawler SD, Povey S, Bagshawe KD. Genetically homozygous choriocarcinoma following pregnancy with hydatidiform mole. Br J Cancer 1988;58:788-92. MEDLINE Abstract

3. Chaganti RSK, Koduru PRK, Chakraborty R, Jones WB. Genetic origin of a trophoblastic choriocarcinoma. Cancer Res 1990;50:6330-3. MEDLINE Abstract

4. Azuma C, Saji F, Nobunaga T, Kamiura S, Kimura T, Tokugawa Y, et al. Studies on the pathogenesis of choriocarcinoma by analysis of restriction fragment length polymorphisms. Cancer Res 1990;50:488-91. MEDLINE Abstract

5. Osada H, Kawata M, Yamada M, Okumura K, Takamizawa H. Genetic identification of pregnancies responsible for choriocarcinomas after multiple pregnancies by restriction fragment length polymorphism analysis. Am J Obstet Gynecol 1991;165:682-8. MEDLINE Abstract

6. Fisher RA, Newlands ES, Jeffreys AJ, Boxer GM, Begent RHJ, Rustin GJS, et al. Gestational and nongestational trophoblastic tumors distinguished by DNA analysis. Cancer 1992;69:839-45. MEDLINE Abstract

7. Suzuki T, Goto S, Nawa A, Kurauchi O, Saito M, Tomoda Y. Identification of the pregnancy responsible for gestational trophoblastic disease by DNA analysis. Obstet Gynecol 1993;82:629-34. MEDLINE Abstract

8. Arima T, Imamura T, Amada S, Tsuneyoshi M, Wake N. Genetic origin of malignant trophoblastic neoplasms. Cancer Genet Cytogenet 1994:73:95-102. MEDLINE Abstract

9. Arima T, Imamura T, Sakuragi N, Higashi M, Kamura T, Fujimoto S, et al. Malignant trophoblastic neoplasms with different modes of origin. Cancer Genet Cytogenet 1995;85:5-15. MEDLINE Abstract

10. Maehara T, Uemura S, Higashi M, Kanazawa K, Unten K. A case of primary gestational ovarian choriocarcinoma. Nippon Sanka Fujinka Gakkai Zasshi 1996;48:427-30 (in Japanese). MEDLINE Abstract

11. Vojtassak J, Repiska V, Zajac V, Konecna B, Korbel M, Danihel L. Origin of choriocarcinoma in previous molar pregnancy proved by DNA analysis. Neoplasia 1996;43:57-9.

12. Japan Society of Obstetrics and Gynecology and the Japanese Pathological Society. The General Rules for Clinical and Pathological Management of Trophoblastic Disease, 2nd ed. Tokyo: Kanehara 1995;8-23.

13. FIGO Annu Rep Results Treat Gynecol Cancer 1991;21:310.

Received May 12, 1999; accepted July 21, 1999
For reprints and all correspondence: Yoshibumi Kaneta, Department of Obstetrics and Gynecology, Social Insurance, Saitama Hospital, 4-9-3 Kita-urawa, Urawa, Saitama 336-0002, Japan

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Last modification: 1 Dec 1999
Copyright© 1999 Foundation for Promotion of Cancer Research.
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