Heterozygous/dispermic complete mole confers a significantly higher risk for post-molar gestational trophoblastic disease.

Hydatidiform moles are classified at the genetic level as androgenetic complete mole and diandric-monogynic partial mole. Conflicting data exist whether heterozygous complete moles are more aggressive clinically than homozygous complete moles. We investigated clinical outcome in a large cohort of hydatidiform moles in Chinese patients with an emphasis on genotypical correlation with post-molar gestational trophoblastic disease. Consecutive products of conceptions undergoing DNA genotyping and p57 immunohistochemistry to rule out molar gestations were included from a 5-year period at Beijing Obstetrics and Gynecology Hospital. Patient demographics and clinical follow-up information were obtained. Post-molar gestational trophoblastic disease or gestational trophoblastic neoplasia was determined by the 2002 WHO/FIGO criteria. A total of 1245 products of conceptions were classified based on genotyping results into 219 complete moles, 250 partial moles, and 776 non-molar gestations. Among 219 complete moles, 186 were homozygous/monospermic and 33 were heterozygous/dispermic. Among 250 partial moles, 246 were triploid dispermic, 2 were triploid monospermic, and 2 were tetraploid heterozygous partial moles. Among 776 non-molar gestations, 644 were diploid without chromosomal aneuploidies detectable by STR genotyping and 132 had various genetic abnormalities including 122 cases of various trisomies, 2 triploid digynic-monoandric non-molar gestations, 7 cases of possible chromosomal monosomy or uniparental disomy. Successful follow-up was achieved in 165 complete moles: post-molar gestational trophoblastic disease developed in 11.6% (16/138 cases) of homozygous complete moles and 37.0% (10/27 cases) of heterozygous complete moles. The difference between the two groups was highly significant (p = 0.0009, chi-square). None of the 218 partial moles and 367 non-molar gestations developed post-molar gestational trophoblastic disease. In conclusion, heterozygous/dispermic complete moles are clinically more aggressive with a significantly higher risk for development of post-molar gestational trophoblastic disease compared with homozygous/monospermic complete moles. Therefore, precise genotyping classification of complete moles is important for clinical prognosis and patient management.

Ovarian hyperstimulation syndrome in a spontaneous pregnancy with invasive mole.

Spontaneous ovarian hyperstimulation syndrome (sOHSS) is an extremely rare event. Herein, we report a case of severe sOHSS with invasive mole in a 29-year-old woman. In this case the full-blown OHSS developed after evacuation when the serum ß-hCG value was declining. Also noticeable was a very high level of cancer antigen-125. Molecular biology study of the follicle-stimulating hormone (FSHR) gene did not detect exonic mutations, but revealed the presence of c.-29G>A (rs1394205) in the 5'-non-coding region of exon 1. The A307T and S680N polymorphisms of exon 10 of FSHR was Thr307 Asn680. Although sOHSS is a rare entity, clinicians must bear the differential diagnosis of sOHSS in mind if a patient presents with gross ascites and other symptoms of ovarian cancer, which also may be signs of OHSS. Whether the single nucleotide polymorphism rs1394205 affects the level of transcriptional activity of the FSHR gene needs to be studied in the future.

Altered p16 and Bcl-2 expression reflects pathologic development in hydatidiform moles and choriocarcinoma.

Abnormal trophoblast differentiation is the main cause of gestational trophoblast diseases in the case of hydatidiform moles and choriocarcinomas. Here we investigated the expression patterns of two gene products, p16 and Bcl-2, implicated in cell cycle regulation and apoptosis, respectively, using immunohistochemistry during normal placenta differentiation, hydatidiform moles (partial, complete and invasive) and post-molar choriocarcinomas. The p16 protein shows a gradual expression in cytotrophoblast of normal villous, from a p16 weak proliferative phenotype to a p16 strong invasive phenotype reaching a maximum around 17 weeks of gestation. The expression pattern in cytotrophoblast was similar in moles in contrast to the villous mesenchyme of invasive moles where p16 was strongly expressed. Bcl-2 expression was syncytiotrophoblast specific in normal placenta and moles and increased gradually during normal differentiation. The results explain the persistence of normal and molar villous fragments during their development and their dramatic invasion in the uterine arteries in case of invasive moles. In choriocarcinomas the weak Bcl-2 expression is associated with weak p16 expression indicating a great apoptotic and proliferative potentials. The results suggest that strong p16 expression in the villous mesenchyme may be responsible in part of the morbidity of the moles, and the key of cancer progression in the choriocarcinomas would be a fast cell-cycle turnover.

Molecular alterations in epidermal growth factor receptors as potential predictors of invasion in complete hydatidiform moles (CHM).

Molecular alterations in Epidermal growth factor receptor (EGFR) were investigated for the first time in molar placenta using protein expression, activation status, differential amplification status and mutational analysis. Invasive lesions showed upregulation of internal domain and downregulation of external domain with concomitantly high gene amplification and phosphorylation. Mutations distributed across different exons in non-invasive cases in contrast to single mutations restricted to exons 4 and 6 in invasive cases displayed a strong correlation to overexpression and phosphorylation status suggesting that higher copies of EGFR gene and mutations in exon 4&6 influence the invasive capacity of trophoblasts and can be used as a biomarker of invasion.

A translocator protein ligand PK11195 shows antigrowth activity in human choriocarcinoma cells.

The potential anticancer agent 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a translocator protein ligand (initially described as a ligand for the peripheral benzodiazepine receptor), induces apoptosis in some lines of human tumor cells. We investigated the effect of PK11195 in the choriocarcinoma cell line, BeWo. BeWo cells were treated with various concentrations of PK11195, and changes in cell growth, the cell cycle, apoptosis, and related parameters were examined. A WST-1 assay showed that BeWo cells were sensitive to the growth inhibitory effect of PK11195. In contrast, the nonsite selective ligand diazepam has a little effect on these cells. Cell cycle analysis indicated that exposure to PK11195 decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine, by the loss of mitochondrial transmembrane potential, and by antibodies directed against histones from fragmented DNA. This induction occurred in conjunction with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. These results suggest that PK11195 may serve as a therapeutic agent for the treatment of choriocarcinoma.

[Function of F10 gene, a novel hydatidiform mole-related gene: a preliminary study].

OBJECTIVE: To study the function of F10 gene, a novel hydaditiform mole-related gene. METHODS: A549 cell line was transfected with the F10 gene of forward or reverse sequence or with the empty vector, respectively. The cellular mRNA was extracted after 24 h of transfection to screen for the differentially expressed genes among the 3 transfected and the control cells using differential display-polymerase chain reaction (ddPCR). RESULTS: The bands representing differentially expressed genes were amplified from the cells, and the products were linked to T-Vector for sequence analysis. Several genes were screened by Blasting and their expressions were confirmed by fluorescent quantitative PCR. CONCLUSION: F10 gene is functionally related to cell proliferation and apoptosis.

[MMP-2/TIMP-2 expression in the trophoblasts of patients with gestational trophoblastic disease].

OBJECTIVE: To explore the role of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of MMP-2 (TIMP-2) in the pathogenesis, development and prognosis of gestational trophoblastic disease (GTD). METHODS: In situ hybridization and immunohistochemistry were utilized for MMP-2/TIMP-2 mRNA and protein detection in normal chorion of women with early gestation, hydatidiform mole, invasive mole, or choricarcinoma. RESULTS: The results revealed that specific staining for mRNA and protein of MMP-2 and the expression of TIMP-2 was reduced in normal chorion of early gestation. In GTD ranging from hydatidiform mole, invasive mole to choricarcinoma, MMP-2 expression tended to increase while TIMP-2 expression underwent an invert change. The positivity rate of MMP-2 and TIMP-2 in gestational trophoblastic tumor group was higher than that of the normal chorion of early gestation group and hydatiform mole group (P<0.05 and P<0.001, respectively). CONCLUSION: A disrupted balance between the activation and inhibition of MMP-2 plays a critical role in the pathogenesis, progression and metastasis of GTD.

Detection of HASH2 (ASCL2) gene expression in gestational trophoblastic disease.

OBJECTIVE: To examine the expression of HASH2 in gestational trophoblastic disease (GTD). STUDY DESIGN: DNA and RNA were isolated from 54 cases of GTD comprising 27 complete hydatidiform moles (CMs), 12 invasive moles (IvMs), 1 placental site trophoblastic tumor (PSTT) and 14 choriocarcinomas (ChCas). Reverse transcriptase polymerase chain reaction and polymerase chain reaction were performed using 2 sets of primers. One pair was used to detect a 190-base pair (bp) segment of the coding region of HASH2 and the other a 68-bp segment of the 3'UTR of the gene that contains a SacII polymorphism. RESULTS: HASH2 was expressed in all normal placenta but not in any of the 27 CMs. In contrast, samples of IvM, PSTT and ChCa, the malignant forms of GTD, all expressed HASH2. Amplification of the 68-bp segment, in the 3'UTR, revealed a product in malignant disease that was larger than 68 bps and resistant to digestion with SacII. CONCLUSION: Negative expression of HASH2 in CM but positive expression in malignant tumors suggests the presence of a specific mechanism for inactivation of the HASH2 gene in CM and reactivation in IvM or ChCa. Based on our data, we speculate that the 3' end of the gene might play an important role in regulating transcription of HASH2.

[Study of gene differential display of hydatidiform moles].

OBJECTIVE: To screen the specific molecular maker of invasive hydatidiform moles (HM) by differential display analysis. METHODS: For dot hybridization, about 1.0 microg of each cDNA sample of invasive and non-invasive HM were labeled as probes using the Dig DNA labeling and Detection Kit (Boehringer Mannheim). The specific expression fragments of invasive HM recovered from PAGE gel were re-amplified by PCR, and the PCR products were dotted onto nylon membrane and hybridized by two probes of invasive and non-invasive HM cDNA respectively. Some fragments with a strong positive hybridization signal were cloned into the polylinker of lasmid PUC19 and were sequenced. The fragments' sequences were searched for homology in the NCBI data using the BLAST (Database: GenBank Human EST entries; Posted date:Aug 31, 2004; Number of letters in database: 1,697,659,032; Number of sequences in database: 3,677,722). RESULTS: The 20 fragments in 28 bands with specific expression in invasive HM were re-amplified, of which 13 showed positive hybridization signals, and 3 were cloned into the polylinker of lasmid PUC19. A fragment in the 3 was a new expressed sequence tag (EST) and the sequence was submitted to NCBI data (dbEST_Id: 10875704; GenBank_Accn: BM403211). CONCLUSION: There are more differences in gene expression between invasive and non-invasive HMs, and differential display analyses are of a potential significance to early diagnosis of invasive HM.

[Association of the novel hydatidiform mole-related gene F10 with the invasiveness of trophoblastic tumor].

OBJECTIVE: To study the expressions of the novel gene F10 associated with hydatidiform mole in different trophoblastic tumors and explore the relation of F10 expression with the invasiveness of malignant trophoblastic tumor. METHODS: In situ hybridization was used to study the expression of F10 in 12 cases of hydatidiform mole, 6 cases of invasive mole, and 8 cases of choriocarcinoma. RESULTS: F10 mRNA was positive in all cases of hydatidiform mole, invasive mole, and choriocarcinoma, and the expression intensity significantly increased in the order of hydatidiform mole, invasive mole and choriocarcinoma (P<0.001). CONCLUSION: The expression of F10 gene may relate to the occurrence and invasiveness of trophoblastic tumor, with possible involvement in the invasion or malignant changes of trophoblastic cells.

Mismatch repair gene promoter methylation and expression in hydatidiform moles.

METHODS: In this study, to investigate the significance of mismatch repair genes (MMR) promoter methylation and expression in the pathogenesis and malignant transformation of hydatidiform moles, we assayed promoter methylation and protein expression of the MMR genes hMLH1 and hMSH2 in gestational trophoblastic diseases (GTDs). DNA was extracted from normal placentas, partial hydatidiform moles, complete hydatidiform moles, and invasive moles, over-digested by methylation-sensitive endonuclease Hpa II, and then the promoters were amplificated by polymerase chain reaction. The protein expression was detected by immunohistochemistry. RESULTS: In the normal placentas, neither hMLH1 nor hMSH2 promoter methylation was detected. Expression of hMLH1 and hMSH2 in cytotrophoblasts was strongly positive. In partial hydatidiform moles and complete hydatidiform moles, hMLH1 and hMSH2 promoter methylation rates were significantly higher than that of normal placentas (P = 0.000), and the protein expression in cytotrophoblasts was significantly lower (P = 0.000). In the invasive moles, hMLH1 and hMSH2 promoter methylation was not significantly different compared with the partial hydatidiform moles and complete hydatidiform moles (P > 0.05). Expression of hMLH1 in the invasive moles (54.5%, 6 out of 11) was not significantly different compared with the partial hydatidiform moles and complete hydatidiform moles (P > 0.05). But hMSH2 expression in the invasive moles (36.5%, 4 out of 11) was weaker than that in complete hydatidiform moles (P = 0.044). Promoter methylation and less expression of hMSH2 were correlated in complete hydatidiform moles (P = 0.001) and invasive moles (P = 0.039). CONCLUSIONS: These results indicated that strong expression of hMLH1 and hMSH2 in the cytotrophoblasts of normal placentas may maintain genome stability. Promoter methylation and down-regulation of the expression of hMLH1 and hMSH2 are probably involved in the pathogenesis of hydatidiform moles.

[Mismatch repair gene promoter methylation and expression in hydatidiform moles and the malignant transformation].

OBJECTIVE: In this study, we assayed promoter hypermethylation and protein expression of the mismatch repair gene (MMR) hMLH1 and hMSH2 in gestational trophoblastic diseases to understand the significance of MMR promoter methylation and expression in the pathogenesis and malignant transformation of hydatidiform mole. METHODS: DNA was extracted from chorion of early pregnancies, partial hydatidiform moles, complete hydatidiform moles, and invasive moles were over digested by methylation sensitive endonuclease Hpa II. Then the promoters were amplificated by polymerase chain reaction. The protein was detected by immunohistochemistry. RESULTS: In the normal placenta, neither hMLH1 nor hMSH2 promoter methylation was detected. Expression of hMLH1 and hMSH2 in cytotrophoblasts was strongly positive, and that was negative or weakly positive in syncytiotrophobasts. In all normal chorion, expression of hMLH1 and hMSH2 in cytotrophoblasts was strongly positive. In partial hydatidiform mole and complete hydatidiform mole, the methylation of hMLH1 and hMSH2 promoters was significantly higher than that of early placenta (P < 0.05), and the protein expression in cytotrophoblasts was significantly lower (P < 0.05). In the invasive mole, hMLH1 and hMSH2 promoter methylation were not significantly different as compared with the partial hydatidiform mole and complete hydatidiform mole (P > 0.05). Expression of hMLH1 in the invasive mole (54.5%, 6/11) was not significantly different as compared with the partial hydatidiform mole and complete hydatidiform mole (P > 0.05). But expression of hMSH2 in the invasive mole (36.4%, 4/11) was weaker than that in complete hydatidiform mole (P = 0.044). Promoter methylation and less expression of hMSH2 had correlations in complete hydatidiform mole or invasive mole. CONCLUSIONS: Strong expressions of hMLH1 and hMSH2 in the cytotrophoblasts of normal placenta may keep the genome stability. Promoter methylation and down-regulation of hMLH1 and hMSH2 are probably involved in the pathogenesis of hydatidiform mole.

The relationship between expression of c-ras, c-erbB-2, nm23, and p53 gene products and development of trophoblastic tumor and their predictive significance for the malignant transformation of complete hydatidiform mole.

OBJECTIVE: The aims of this retrospective study by means of immunohistochemical staining were (1) to study the expression of c-ras, c-erbB-2, p53, and nm23 gene products in complete hydatidiform moles that progress to gestational trophoblastic tumor and in those that remit spontaneously after evacuation, and (2) to estimate the predictive value of the expression of these four gene products in malignant transformation of complete hydatidiform mole. METHODS: Clinical data of patients with complete hydatidiform mole were obtained by retrospective chart review. Formalin-fixed paraffin sections of 50 cases of complete mole that progressed to gestational tumor and 32 cases of complete mole that remitted spontaneously were studied immunohistochemically for c-ras, c-erbB-2, p53, and nm23 proteins. The prognostic value of the proteins for the malignant transformation of complete mole was analyzed by multiple logistic regression and stepwise logistic estimation. Sections of 30 cases of invasive mole and 19 cases of choriocarcinoma were also immunohistologically studied for expression of the proteins. RESULTS: Expression of c-erbB-2 and p53 gene products was significantly increased and expression of nm23 and c-ras products was remarkably decreased in complete hydatidiform moles that progressed into postmolar tumor compared with those that remitted spontaneously after evacuation. There was no significant difference in the expression of the four genes in invasive mole and in choriocarcinoma. A logistic estimation model for predicting malignant transformation of complete mole was established based on the expression of gene products. When the expression of four gene products was used, the predictive sensitivity of the regression model was 86.0%, and the specificity was 75.0%. The positive predictive value was 84.3%, the negative predictive value was 77.4%. Logistic stepwise regression analysis showed that the altered expression of c-erbB-2 and nm23 products had strong predictive value, while the expression of c-ras and p53 products had no significant predictive value for the malignant transformation of complete mole. CONCLUSION: The altered expression of c-ras, c-erbB-2, nm23, and p53 gene products may be important in the pathogenesis of gestational trophoblastic tumor. The decreased expression of nm23 protein and increased expression of c-erbB-2 protein are strong predictors for the malignant transformation of complete mole.

Current understandings of the molecular genetics of gestational trophoblastic diseases.

Gestational trophoblastic disease (GTD) is a heterogeneous group of diseases characterized by abnormally proliferating trophoblastic tissues. This includes partial and complete hydatidiform moles, invasive mole, choriocarcinoma and placental site trophoblastic tumour. Cytogenetic studies revealed that hydatidiform moles contain either solely (as in complete moles) or an excess (as in partial moles) of paternal contribution to the genome. Genomic imprinting is believed to play a pivotal role in the pathogenesis of hydatidiform moles. However its precise role and mechanism remains poorly understood. Hydatidiform mole carries a potential of malignant transformation. Similar to other human cancers, malignant transformation in gestational trophoblastic tumours is likely a multistep process and involves multiple genetic alterations including activation of oncogenes and inactivation of tumour suppressor genes. In addition, expression of telomerase activity, altered expression of cell--cell adhesion molecules and abnormal expression of matrix metalloproteinases have also been reported in GTD. These represent disruption of the delicate balance and regulation of cellular processes including proliferation, differentiation, apoptosis and invasion. The significance of these alterations in the pathogenesis and malignant transformation of gestational trophoblastic diseases is reviewed in this paper.

Mola invasiva--special form of GTD.

Invasive hydatidiform mole is a relative rare form of gestational trophoblastic disease (GTD). Most of hydatidiform moles remit after evacuation but some of them have the tendency to invade the myometrium. In some rare cases the trophoblastic tissue can be found in other tissues like lungs, vulva, vagina or broad ligament. The aim of the study was to demonstrate some of clinical, immunohistochemical and DNA analysis findings of a patient with a previous diagnosis of a complete hydatidiform mole.

DOC-2/hDab2, a candidate tumor suppressor gene involved in the development of gestational trophoblastic diseases.

Gestational trophoblastic diseases comprise a spectrum of interrelated diseases including partial mole, complete mole and gestational choriocarcinoma. Using reverse transcriptase PCR (RT-PCR) analysis, we identified higher levels of DOC-2/hDab2 expression in the normal trophoblast cells in culture than in choriocarcinoma cell lines. Subsequent study using immunohistochemistry showed high levels of DOC-2/hDab2 protein expression in normal trophoblast tissues but significantly lower levels of expression in gestational trophoblastic disease tissues, particularly in complete mole and choriocarcinoma. When DOC-2/hDab2 was transfected into the choriocarcinoma cell lines, Jar, JEG and BeWo, the stable transfectants showed significantly reduced growth rate in culture. These data suggest that down regulation of DOC-2/hDab2 may play an important role in the development of gestational trophoblastic diseases.

DNA polymorphism analysis of a case of complete hydatidiform mole coexisting with a fetus.

A complete hydatidiform mole coexisting with a fetus is a rare condition. The diagnosis is often difficult because of the morphological similarity to a partial mole, but is crucial to management in the postmolar course. We present a case of molar pregnancy coexisting with a fetus in which DNA polymorphism analysis revealed a different genetic origin for the fetal and molar parts. This is the only known case of a complete mole in a twin pregnancy complicated by pre-eclampsia followed by maternal pulmonary oedema. During follow-up, the patient developed a clinically invasive mole which was successfully treated with chemotherapy. In this case, genetic analysis unequivocally diagnosed a twin pregnancy consisting of a complete hydatidiform mole and a fetus.

Lack of mutation in tumour-suppressor gene p53 in gestational trophoblastic tumours.

The objectives of this study were to better our understanding of the carcinogenesis of gestational trophoblastic tumours and to investigate the possible presence of mutational alteration of the p53 tumour-suppressor gene in these tumours. Amplification-based direct DNA sequencing was performed on 14 hydatidiform moles, six invasive moles, eight choriocarcinomas and ten normal early placental tissues. No mutation in exons 5-8 was detected in any of these 38 tissue specimens. These results suggest that a mutation in p53 tumour suppressor either does not exist or is a very rare event in gestational trophoblastic tumours. The gestational trophoblastic tumours probably involve a tumour-suppressor gene other than p53 gene or may follow a completely different pathway to their malignant phenotype.