—  SYMPOSIUM #41  —

Gestational Trophoblastic Disease
Moderator: Dr. le-Ming Shih

Section 6 - Application of New Laboratory Techniques in Diagnosis and Prognosis Prediction of Molar Lesions

Annie NY Cheung
Department of Pathology, Queen Mary Hospital
The University of Hong Kong
Hong Kong


In recent years, advances in laboratory techniques have enabled more elaborate biological studies of gestational trophoblastic disease [1] . It has been highlighted that histological distinction between hydropic abortion and partial mole and between complete and partial moles, especially at early gestational age, may be difficult. Moreover, few parameters have been found useful in predicting the clinical progress of hydatidiform mole to persistent gestational trophoblastic neoplasia which requires chemotherapy [2] . Similar to other human cancers, malignant transformation in gestational trophoblastic tumors is likely a multistep process involving activation of oncogenes and inactivation of tumor suppressor genes. In addition, disruption of the delicate regulation of cellular processes including proliferation, differentiation, apoptosis and invasion, as well as expression of telomerase activity, may also be important mechanisms in determining behavior of these diseases. The application of these techniques on such processes as attempts to improve diagnosis and management of gestational trophoblastic disease will be discussed.

Cytogenetic Analysis
Hydatidiform mole is classified into partial and complete subtypes according to histopathological and genetic criteria [3, 4] . Genetically, a complete mole is diploid without maternal contribution, whereas a partial mole is triploid with a maternal chromosome complement [5] . Various diagnostic tools have been considered useful for such distinction including cytogenetic analysis, DNA flow cytometry, chromosome in situ hybridization and PCR based genotyping [6, 7, 8, 9, 10] .

Fluorescent microsatellite genotyping to detect the presence or absence of maternal genome in a hydatidiform mole and chromosome in situ hybridization to analyze the ploidy have been performed to correlate with histological diagnosis [11] . The genotyping results correlated with chromosome in situ hybridization findings in all cases, i.e. triploid hydatidiform moles had maternal-derived alleles, while diploid hydatidiform moles were purely androgenetic. Compared with genetic diagnosis, histological evaluation was more reliable for the diagnosis of a complete mole than that of a partial mole. Genotyping and chromosome in situ hybridization can thus provide reliable adjunct to histology for the classification of a hydatidiform mole.

As a partial mole still carries a risk of developing gestational trophoblastic neoplasia, follow-up is considered necessary for both complete and partial moles. Actually, among the six histologically diagnosed partial hydatidiform mole which subsequently metastasized, four cases had a diploid karyotype and no maternal alleles. These cases were thus actually partial hydatidiform mole [12] . However, it seems that there was no correlation between the presence of a Y chromosome and the development of persistent gestational trophoblastic disease with or without metastasis [13, 14]

Telomerase Activity
Telomerase is a ribonucleoprotein complex that is thought to add telomeric DNA repeats onto the ends of chromosomes during cell proliferation. Since cells of most normal tissues lack this activity, successive cell division will result in progressive shortening of telomeres. It is hypothesised that telomere shortening both in vivo and in vitro is the mitotic clock that determines the onset of cellular senescence. It is also suggested that activation of telomerase is necessary for the sustained proliferation and development of tumours [15, 16] . Telomerase activity in hydatidiform mole was assessed and compared with normal placentas of different gestational age and choriocarcinoma using the telomeric repeat amplification protocol (TRAP) assay. Telomerase activation was demonstrated in hydatidiform mole. Hydatidiform mole, which subsequently developed persistent disease, especially those which metastasised, were more likely to express telomerase activity [17] . Thus, assessment of telomerase activity in hydatidiform mole may be useful in the clinical management.

Proliferative and Apoptotic Activities
The role of assessing proliferative activity by immunohistochemistry for the proliferating cell nuclear antigen (PCNA), Ki-67, m inichromosome maintenance protein mcm7 and helix-loop-helix Id proteins (inhibitor of differentiation or inhibitor of DNA binding) , as a measure to predict the clinical progress of hydatidiform mole have been assessed. There was no statistically significant difference in such proliferative index between those patients who developed persistent disease and those who did not [18, 19, 20, 21] . The proliferation index is probably not useful in predicting the prognosis of molar pregnancies. On the other hand, proliferation index assessed by the newer generation of cell cycle markers such as MCM7 and Id was significantly higher in partial hydatidiform mole and complete hydatidiform mole than in spontaneous abortion and may thus be useful to distinguish hydropic abortion from early hydatidiform mole [20, 21] .

More promising results were obtained from studies on apoptotic activity in GTD. The apoptotic activity was initially studied by the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) method [22] . Apoptotic activity were significantly different among normal placenta, spontaneous abortion, choriocarcinoma and hydatidiform mole. Apoptotic indices of those hydatidiform moles that spontaneously regressed was statistically higher than those that developed persistent gestational trophoblastic neoplasia. A poptotic activity of trophoblastic lesions in general inversely correlated with Bcl-2 expression but no t Bax expression. A poptotic index may thus be a useful prognostic marker for clinical progress of hydatidiform mole . Bcl-2 expression is probably regulating apoptosis in trophoblastic lesions while Bax expression is not. Such findings were further confirmed when apoptotic activity of gestational trophoblastic disease was further assessed using the M30 CytoDeath antibody [23] . Apoptotic indices assessed by M30 antibody and TUNEL correlated with each other.

Both the TUNEL method and M30 immunohistochemistry are techniques that can be conveniently applied in routine surgical pathology and provide useful information for prediction of clinical progress of patients.

Differential Expression Display Studies
Recent advances in molecular methodology have facilitated high-throughput analysis of differential gene expression in GTD. cDNA array is available from commercial companies and human genome centers allowing simultaneous analysis of various genes known to be involved in human carcinogenesis, cell cycle and transcription regulation, DNA repair and synthesis, apoptosis, growth factors and receptor signaling [24] . The differential expression profile of genes with known functions can then be identified leading to better understanding of the disease [25] . On the other hand, Suppression Subtractive Hybridization (SSH) technique, which combines both the suppression PCR amplification and cDNA subtraction, is sensitive and efficient for generating cDNA highly enriched for differentially expressed genes with both high and low abundance leading to generation of two subtractive libraries [26] . These may include novel transcripts with uncharacterized functions.

These approaches have been utilized to study gestational trophoblastic disease from two angles. Firstly, differentially expressed genes can be identified in hydatidiform mole related to different clinical outcome, i.e. spontaneous regression and developing persistent gestational trophoblastic neoplasia . Such genes may be useful marker for predicting progress of hydatidiform mole . Secondly, differentially expressed genes in hydatidiform mole as compared with non-molar placentas can be identified and these genes may contribute toward understanding the pathogenesis of complete hydatidiform mole and may be explored as markers for diagnosis of hydatidiform mole in histologically difficult cases.

HM That Developed GTN Vs Spontaneously Regressed HM
Due to earlier findings that low apoptotic activity correlated with the progression of hydatidiform mole to gestational trophoblastic neoplasia , apoptosis-related genes may determine this progression. The differential expression of apoptotic genes in hydatidiform mole s that subsequently developed into gestational trophoblastic neoplasia was compared with hydatidiform mole s that spontaneously regressed using a human apoptosis array. Increased expression of Mcl-1, an antiapoptotic gene, was detected in hydatidiform mole s that subsequently developed into gestational trophoblastic neoplasia [27] . It was confirmed by follow up quantitative real time PCR and protein expression analysis. Moreover, Mcl-1 immunoreactivity, which was detected predominantly in cytotrophoblasts, was correlated with the apoptotic index, assessed with M30 cytoDeath immunohistochemistry.

Tissue-specific chips were constructed from the subtracted cDNA libraries comparing the differential expression pattern of genotyped hydatidiform mole that spontaneously regressed and that subsequently developed metastatic gestational trophoblastic neoplasia , followed by cDNA microarray analysis. Among the differentially expressed transcripts identified, quantitative RNA analysis confirmed down-regulation of ferritin light polypeptide (FTL) and insulin-like growth factor binding protein 1 (IGFBP1) in hydatidiform mole that subsequently developed gestational trophoblastic neoplasia when compared with those hydatidiform mole that regressed [28] . Immunohistochemical analysis further confirmed reduced IGFBP1 protein expression in hydatidiform mole that developed gestational trophoblastic neoplasia . Our findings suggested that reduced expression of genes related to cell invasion and immunosuppression, especially FTL and IGFBP1, were associated with development of gestational trophoblastic neoplasia , and this finding may provide a better understanding of the pathogenesis of gestational trophoblastic neoplasia . The potential application of FTL and IGFBP1 in management of patients with hydatidiform mole should be explored.

GTD Vs Non-Molar Trophoblasts
Using cDNA array, Vegh et al has demonstrated down-regulation of Hsp-27 in choriocarcinoma cell lines suggesting that it may contribute to the extreme sensitivity of trophoblastic tumors to chemotherapy [29] . Besides confirming such findings, our group employing cDNA array hybridization to compare gene expression profiles in choriocarcinoma cell lines (JAR, JEG, and BeWo) and normal first trimester human placentas, has also identified differential expression of Caspase 10 and its closely related family member caspase 8 in choriocarcinoma [30] . Down-regulation of caspase 10 in choriocarcinoma was detected by both human cDNA expression array and human 1.2 array. Caspase 10 mRNA expression was significantly lower in hydatidiform mole and choriocarcinoma compared with normal placenta. The caspase 8 and 10 proteins were expressed predominantly in the cytotrophoblast and syncytiotrophoblast, respectively, with significantly lower expression in choriocarcinomas than other trophoblastic tissues. Immunoreactivity for both caspase 8 and 10 correlated with the apoptotic index previously assessed by TUNEL and M30 approaches. These results suggest that the downregulation of capases 8 and 10 might contribute to the pathogenesis of choriocarcinoma.

As an attempt to assess the molecular pathogenesis of complete hydatidiform mole , suppression subtractive hybridization (SSH) combined with cDNA microarray was used to compare the gene expression pattern of complete hydatidiform mole compared with normal first-trimester placenta of similar gestational ages [31] . cDNA microarray analysis using tissue-specific chips constructed with subtracted cDNA libraries identified 13 differentially expressed gene transcripts. Quantitative real-time PCR confirmed up-regulation of human chorionic gonadotropin beta subunit (CGB) and KIAA1200, a G-protein regulator, as well as down-regulation of osteopontin (SPP1) in genotyped complete hydatidiform mole when compared with normal placentas. Down-regulation of osteopontin has been previously reported by Batorfi et al from a candidate gene approach [32] . These candidate genes may contribute toward understanding the mechanism involved with the development and progression of complete hydatidiform mole .

Epigenetic Downregulation of Tumor Suppressor Genes
While promoter hypermethylation has recently been found to be an important epigenetic mechanism causing gene inactivation, the methylation status of genes in hydatidiform mole and choriocarcinoma and its significance is relatively unexplored. The methylation status of the promoter regions of six genes, p16, HIC-1, TIMP3, GSTP1, death-associated protein kinase (DAPK), and E-cadherin in hydatidiform moles, choriocarcinomas, and first trimester placenta have recently been studied by methylation-specific PCR in correlation with protein and quantitative real-time RT-PCR studies. Among the six genes examined, the promoter region of four genes (E-cadherin, HIC-1, p16, TIMP3) in choriocarcinoma and three genes (E-cadherin, HIC-1, p16) in hydatidiform mole exhibited aberrant methylation whereas none was hypermethylated in normal placenta. Treatment with a demethylation drug, 5-aza-2'-deoxycytidine, in choriocarcinoma cell lines restored TIMP3 expression confirming that promoter methylation of TIMP3 is involved in suppression of TIMP3 expression. [33] . There was a significant correlation between methylation and reduced expression of p16, E-cadherin, and TIMP3. Moreover, promoter hypermethylation of p16 alone, or combined with E-cadherin, was significantly correlated to development of gestational trophoblastic neoplasia . Thus, hypermethylation of multiple genes, especially p16, might be related to the subsequent development of gestational trophoblastic neoplasia [34] .

Conclusions
With recent advances in molecular techniques, molecular factors in the pathogenesis of gestational trophoblastic disease have been studied. Yet, further works are still necessary to provide a better understanding of this peculiar group of disease, which may not only provide important insights into its pathogenesis, but also useful prognostic indicators which may guide therapy.

References:
  1. Li, H. W., Tsao, S. W., and Cheung, A. N. Current understandings of the molecular genetics of gestational trophoblastic diseases. Placenta, 23: 20-31, 2002.

  2. Cheung, A. N. Pathology of gestational trophoblastic diseases. Best Pract Res Clin Obstet Gynaecol, 17: 849-868, 2003.

  3. Szulman, A. E. and Surti, U. The syndromes of hydatidiform mole. II. Morphologic evolution of the complete and partial mole. Am J Obstet Gynecol, 132: 20-27, 1978.

  4. Szulman, A. E. and Surti, U. The syndromes of hydatidiform mole. I. Cytogenetic and morphologic correlations. Am J Obstet Gynecol, 131: 665-671, 1978.

  5. Kajii, T. and Ohama, K. Androgenetic origin of hydatidiform mole. Nature, 268: 633-634, 1977.

  6. Fisher, R. A. and Newlands, E. S. Rapid diagnosis and classification of hydatidiform moles with polymerase chain reaction. Am J Obstet Gynecol, 168: 563-569, 1993.

  7. Hemming, J. D., Quirke, P., Womack, C., Wells, M., Elston, C. W., and Bird, C. C. Diagnosis of molar pregnancy and persistent trophoblastic disease by flow cytometry. J Clin Pathol, 40: 615-620, 1987.

  8. Lage, J. M., Driscoll, S. G., Yavner, D. L., Olivier, A. P., Mark, S. D., and Weinberg, D. S. Hydatidiform moles. Application of flow cytometry in diagnosis. Am J Clin Pathol, 89: 596-600, 1988.

  9. Paradinas, F. J., Browne, P., Fisher, R. A., Foskett, M., Bagshawe, K. D., and Newlands, E. A clinical, histopathological and flow cytometric study of 149 complete moles, 146 partial moles and 107 non-molar hydropic abortions. Histopathology, 28: 101-110, 1996.

  10. Van de Kaa, C. A., Hanselaar, A. G., Hopman, A. H., Nelson, K. A., Peperkamp, A. R., Gemmink, J. H., Beck, J. L., De Wilde, P. C., Ramaekers, F. C., and Vooijs, G. P. DNA cytometric and interphase cytogenetic analyses of paraffin-embedded hydatidiform moles and hydropic abortions. J Pathol, 170: 229-238, 1993.

  11. Lai, C. Y., Chan, K. Y., Khoo, U. S., Ngan, H. Y., Xue, W. C., Chiu, P. M., Tsao, S. W., and Cheung, A. N. Analysis of gestational trophoblastic disease by genotyping and chromosome in situ hybridization. Mod Pathol, 17: 40-48, 2004.

  12. Cheung, A. N., Khoo, U. S., Lai, C. Y., Chan, K. Y., Xue, W. C., Cheng, D. K., Chiu, P. M., Tsao, S. W., and Ngan, H. Y. Metastatic trophoblastic disease after an initial diagnosis of partial hydatidiform mole: genotyping and chromosome in situ hybridization analysis. Cancer, 100: 1411-1417, 2004.

  13. Cheung, A. N., Sit, A. S., Chung, L. P., Ngan, H. Y., O'Hanlan, K., Wong, L. C., and Ma, H. K. Detection of heterozygous XY complete hydatidiform mole by chromosome in situ hybridization. Gynecol Oncol, 55: 386-392, 1994.

  14. Mutter, G. L., Pomponio, R. J., Berkowitz, R. S., and Genest, D. R. Sex chromosome composition of complete hydatidiform moles: relationship to metastasis. Am J Obstet Gynecol, 168: 1547-1551, 1993.

  15. Feng, J., Funk, W. D., Wang, S. S., Weinrich, S. L., Avilion, A. A., Chiu, C. P., Adams, R. R., Chang, E., Allsopp, R. C., Yu, J., and et al. The RNA component of human telomerase. Science, 269: 1236-1241, 1995.

  16. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L., Coviello, G. M., Wright, W. E., Weinrich, S. L., and Shay, J. W. Specific association of human telomerase activity with immortal cells and cancer. Science, 266: 2011-2015, 1994.

  17. Cheung, A. N., Zhang, D. K., Liu, Y., Ngan, H. Y., Shen, D. H., and Tsao, S. W. Telomerase activity in gestational trophoblastic disease. J Clin Pathol, 52: 588-592, 1999.

  18. Cheung, A. N., Ngan, H. Y., Chen, W. Z., Loke, S. L., and Collins, R. J. The significance of proliferating cell nuclear antigen in human trophoblastic disease: an immunohistochemical study. Histopathology, 22: 565-568, 1993.

  19. Cheung, A. N., Ngan, H. Y., Collins, R. J., and Wong, Y. L. Assessment of cell proliferation in hydatidiform mole using monoclonal antibody MIB1 to Ki-67 antigen. J Clin Pathol, 47: 601-604, 1994.

  20. Xue, W. C., Feng, H. C., Chan, K. Y., Chiu, P. M., Ngan, H. Y., Khoo, U. S., Tsao, S. W., Chan, K. W., and Cheung, A. N. Id helix-loop-helix proteins are differentially expressed in gestational trophoblastic disease. Histopathology, 47: 303-309, 2005.

  21. Xue, W. C., Khoo, U. S., Ngan, H. Y., Chan, K. Y., Chiu, P. M., Tsao, S. W., and Cheung, A. N. Minichromosome maintenance protein 7 expression in gestational trophoblastic disease: correlation with Ki67, PCNA and clinicopathological parameters. Histopathology, 43: 485-490, 2003.

  22. Wong, S. Y., Ngan, H. Y., Chan, C. C., and Cheung, A. N. Apoptosis in gestational trophoblastic disease is correlated with clinical outcome and Bcl-2 expression but not Bax expression. Mod Pathol, 12: 1025-1033, 1999.

  23. Chiu, P. M., Ngan, Y. S., Khoo, U. S., and Cheung, A. N. Apoptotic activity in gestational trophoblastic disease correlates with clinical outcome: assessment by the caspase-related M30 CytoDeath antibody. Histopathology, 38: 243-249, 2001.

  24. Zhao, N., Hashida, H., Takahashi, N., Misumi, Y., and Sakaki, Y. High-density cDNA filter analysis: a novel approach for large-scale, quantitative analysis of gene expression. Gene, 156: 207-213, 1995.

  25. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y., Su, Y. A., and Trent, J. M. Use of a cDNA microarray to analyse gene expression patterns in human cancer. Nat Genet, 14: 457-460, 1996.

  26. Diatchenko, L., Lau, Y. F., Campbell, A. P., Chenchik, A., Moqadam, F., Huang, B., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E. D., and Siebert, P. D. Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A, 93: 6025-6030, 1996.

  27. Fong, P. Y., Xue, W. C., Ngan, H. Y., Chan, K. Y., Khoo, U. S., Tsao, S. W., Chiu, P. M., Man, L. S., and Cheung, A. N. Mcl-1 expression in gestational trophoblastic disease correlates with clinical outcome: a differential expression study. Cancer, 103: 268-276, 2005.

  28. Feng, H. C., Tsao, S. W., Ngan, H. Y., Xue, W. C., Chiu, P. M., and Cheung, A. N. Differential expression of insulin-like growth factor binding protein 1 and ferritin light polypeptide in gestational trophoblastic neoplasia: combined cDNA suppression subtractive hybridization and microarray study. Cancer, 104: 2409-2416, 2005.

  29. Vegh, G. L., Fulop, V., Liu, Y., Ng, S. W., Tuncer, Z. S., Genest, D. R., Paldi-Haris, P., Foldi, J., Mok, S. C., and Berkowitz, R. S. Differential gene expression pattern between normal human trophoblast and choriocarcinoma cell lines: downregulation of heat shock protein-27 in choriocarcinoma in vitro and in vivo. Gynecol Oncol, 75: 391-396, 1999.

  30. Fong, P. Y., Xue, W. C., Ngan, H. Y., Chiu, P. M., Chan, K. Y., Tsao, S. W., and Cheung, A. N. Caspase activity is downregulated in choriocarcinoma: a cDNA array differential expression study. J Clin Pathol, 59: 179-183, 2006.

  31. Feng, H. C., Tsao, S. W., Ngan, H. Y., Kwan, H. S., Shih, S. M., Xue, W. C., Chiu, P. M., Chan, K. W., and Cheung, A. N. Differential gene expression identified in complete hydatidiform mole by combining suppression subtractive hybridization and cDNA microarray. Placenta, 27: 521-526, 2006.

  32. Batorfi, J., Fulop, V., Kim, J. H., Genest, D. R., Doszpod, J., Mok, S. C., and Berkowitz, R. S. Osteopontin is down-regulated in hydatidiform mole. Gynecol Oncol, 89: 134-139, 2003.

  33. Feng, H., Cheung, A. N., Xue, W. C., Wang, Y., Wang, X., Fu, S., Wang, Q., Ngan, H. Y., and Tsao, S. W. Down-regulation and promoter methylation of tissue inhibitor of metalloproteinase 3 in choriocarcinoma. Gynecol Oncol, 94: 375-382, 2004.

  34. Xue, W. C., Chan, K. Y., Feng, H. C., Chiu, P. M., Ngan, H. Y., Tsao, S. W., and Cheung, A. N. Promoter hypermethylation of multiple genes in hydatidiform mole and choriocarcinoma. J Mol Diagn, 6: 326-334, 2004.