Hydatidiform mole

* Context.-Because there are differences in the origin, morphology, and natural history of hydropic placental villous issues, it is important to identify and document rare specimens that deviate from the diploid complete hydatidiform mole (CM), triploid partial hydatidiform mole (PM), or diploid hydropic abortion (HA). Tetraploid hydropic placentas have rarely been studied.

Objectives.-To evaluate the frequency of p57^sup Kip2^ protein (p57) expression in tetraploid hydropic placentas and to determine its clinicopathologic significance.

Design.-Forty hydropic DNA tetraploid placental specimens were evaluated by immunohistochemistry of formalin-fixed tissues, using a monoclonal antibody against p57, a putative paternally imprinted inhibitor gene. DNA ploidy in all cases was analyzed by flow cytometry.

Results.-Thirty cases were histologically diagnosed as CMs, 10 were HAs, and none were PMs. In all HAs, nuclear p57 was strongly expressed in cytotrophoblasts, intermediate trophoblasts, and villous stromal cells. In contrast, in CMs, p57 expression in cytotrophoblasts and villous stromal cells was either absent (26 cases) or very low (4 cases). Assuming that the degree of molar change roughly correlates with the proportion of paternal chromosomes present, all chromosomes might be paternally derived in all tetraploid CMs and the 10 HAs, including 2 that were karyotyped as 92,XXYY or 90,XXYY,-13,-14, which were presumably due to 2 sets of chromosomes each from paternal and maternal origin.

Conclusions.-Expression of p57 is aberrant in tetraploid CMs. This finding is in line with the hypothesis that the loss of p57 is involved in the abnormal development of androgenetic CMs. For the evaluation of a patient with trophoblastic disease, p57 immunostaining is an ancillary diagnostic method that may be used in concert with flow cytometry.

(Arch Pathol Lab Med. 2004;128:897-900)

Hydropic placental villous tissues represent a constellation of complete hydatidiform mole (CM), partial hydatidiform mole (PM), and hydropic abortion (HA). Cytogenetic studies have identified marked differences in the genetic makeup of molar placentas. A CM is diploid, generally has a 46,XX or a 46,XY karyotype, and is entirely androgenetically derived.1 A PM usually contains an extra haploid set of paternally derived DNA, and therefore has 9 69 chromosomes (termed triploid).2 Persistent gestational trophoblastic disease follows PM in approximately 5% of cases, whereas up to 20% of CM cases will require chemotherapy.3 Because there are differences in the origin, morphology, and natural history of hydatidiform mole or HA inferred by histologie classification, it is important to identify and document rare specimens that deviate from the diploid CM, triploid PM, or diploid HA. Tetraploid hydropic placental villous tissues have been reported only in small series, and there have been few comprehensive studies.4-6

The cyclin-dependent kinase inhibitor p57^sup Kip2^ protein (p57) recently has been included among the paternally imprinted genes in humans.7 p57 has a restricted tissue distribution with high levels of expression in human placenta, suggesting that it has a specialized function in cell cycle control during implantation and placental development.8-10 This article presents the results of a study of immunohistochemical detection of p57 expression in patients with DNA tetraploid hydropic placentas; the objectives were to evaluate the frequency of expression and to determine its clinicopathologic significance.

MATERIALS AND METHODS

Forty specimens of placental material from abortions in which swelling of villi was macroscopically or microscopically observed and that were tetraploid by flow cytometry were collected from 1978 through 2001. All materials were obtained by curettage or hysterectomy at the Jikei University Hospital (Tokyo, Japan) or its affiliated hospitals. Patient age ranged from 18 to 52 years (mean age, 29.3 years) and gestational age ranged from 6 to 14 weeks (mean, 10.3 weeks). As controls, 10 cases of voluntary artificial abortions during the first trimester (gestational age range, 6-12 weeks) that were DNA diploid were used. Part of this study has been reported previously.5,11,12

Routine 4-µm sections of all specimens (1-5 blocks from each case) stained with hematoxylin-eosin were reviewed. The diagnosis of molar pregnancy was based on the pathologic criteria of Szulman and Surti2; namely, the diagnosis of CM was made when there was complete hydatidiform change from edema to central cistern formation, absence of an embryo, and conspicuous trophoblastic hyperplasia. The diagnosis of PM was made in the presence of partial villous involvement (normal and edematous villi), an embryo or fetus, mild to moderate focal trophoblastic hyperplasia, and "trophoblastic inclusion." Trophoblastic hyperplasia is an essential feature in differentiating hydatidiform mole from simple HA.

Immunohistochemical studies were performed using archival 4-µm, formalin-fixed, paraffin-embedded sections. Expression of p57 is assessed by immunohistochemical examination using antihuman p57 monoclonal antibody (2-A µg/mL; Lab Vision, Fremont, Calif). After blocking of endogenous peroxidase activity, the sections were autoclaved in 10mM citrate buffer, pH 6, for 10 minutes at 132°C for antigen retrieval. The avidin-biotin peroxidase complex technique was used, and the sections were incubated for 30 minutes at room temperature. Appropriate positive and negative control experiments were also performed for the immunohistochemical study. Cells were regarded as p57-positive only when distinct nuclear immunostaining was revealed, excluding cells with faint or weak staining or cytoplasmic granular deposits.

The ploidy status was obtained by flow cytometry. Flow cytometric DNA analysis was performed on formalin-fixed, paraffin-embedded tissue blocks using the technique of Hedley et al.13 A placenta was classified as tetraploid if the peak in the G2/M region represented more than 25% of the cells and was followed by an octaploid population and if the DNA index was between 1.90 and 2.10. Karyotypic analysis was also performed on 2 cases (one was 92,XXYY and the other was 90,XXYY,-13,-14; gestational ages were 8 and 10 weeks, respectively).

Follow-up after molar evacuation included urine [beta]-subunit human chorionic gonadotropin (HCG) titers measured every week until 12 weeks or serum HCG titers measured every week until negarive. A diagnosis of persistent trophoblastic disease was determined by inappropriate falling urine HCG, rising serum HCG, or plateau of previously falling HCG based on 3 consecutive titers during the follow-up after molar evacuation.

The Fisher exact test was used for comparison of groups. A result was considered statistically significant at P

RESULTS

Among 40 tetraploid hydropic placentas, 30 were diagnosed histologically as CM, 10 were HA, and none were PM. Placentas with 92,XXYY and 90,XXYY,-13,-14 karyotypes represented HAs. The gestational ages of CMs and HAs were between 8 and 14 weeks, and between 6 and 12 weeks, respectively.

The results of p57 expression are summarized in the Table. In all control placental tissue, nuclear p57 was strongly expressed in cytotrophoblasts, villous and extravillous (placental site) intermediate trophoblasts, villous stromal cells, and decidual stromal cells, but was uniformly absent in syncytiotrophoblasts (Figure 1). Gestational endometrial glands were negative. p57 expression in cytotrophoblasts and villous stromal cells was either absent (26 cases) or very low (1%-10% positive cells) (4 cases) in the tetraploid CMs (Figure 2). Villous or extravillous intermediate trophoblasts stained for p57 in 8 cases of tetraploid CM. The results of all HAs were comparable to those of the control placental tissues (Figure 3). There was a significant difference in p57 expression in cytotrophoblast and villous stromal cells between tetraploid CMs and tetraploid HAs (P = .002) and between tetraploid CMs and control placentas (P = .002), but there was no significant difference between tetraploid HAs and control placentas. Decidual stromal cells were positive for p57 in all cases examined and provided a reliable internal control (Figure 4).

Follow-up data were available for 18 patients with tetraploid CM. Fourteen patients (78%) experienced spontaneous resolution, and 4 cases (22%) were complicated by persistent disease. Invasive mole was identified on hysterectomy materials from 3 patients, and 1 patient developed choriocarcinoma, which had a diploid DNA content. There was no significant difference of histology between tetraploid CMs with and without persistent disease. p57 expression was negative in 17 CMs without persistent disease, and 1 CM without persistent disease expressed p57 (1%-10% positive cells ). p57 expression was absent in all 4 CMs with persistent disease. No significant difference was seen in p57 expression between tetraploid CMs with and without persistent disease (P = .60).

COMMENT

In the present study, expression of p57 (a genomically imprinted cyclin-dependent kinase inhibitor and a paternally imprinted, maternally expressed gene) was observed at high frequency in normal placentas and tetraploid HAs, but was absent or present at low frequency in tetraploid CMs. These findings were compatible with those of diploid HAs and diploid CMs in my previous study.12 The results support the hypothesis that aberrant expression of p57 is involved in the abnormal development of androgenetic tetraploid CMs, which arise from fertilization of empty eggs. p57 is a cell cycle inhibitor and tumor suppressor, and lack of p57 activity can lead to loss of cell cycle control and hyperproliferation.14 Thus, low or absent p57 expression likely plays a role in the abnormal proliferation and differentiation of trophoblasts in tetraploid CMs. The phenomenon also may be related to the increased number of villous stromal cells in early CMs.15 The lack of p57 expression in syncytiotrophoblasts in all specimens examined may indicate that p57 is not directly related to syncytiotrophoblasts, which are thought to be terminally differentiated and nonproliferating cells.12

In both CMs and PMs, the presence of 2 paternal chromosome sets is associated with hyperplasia of trophoblasts; a triploid conceptus with 1 paternal and 2 maternal sets is not usually associated with a pathologic diagnosis of PM.16 With rare exception, the degree of molar change and the risk of persistent disease roughly correlate with the proportion of paternal chromosomes present.17,18 Chromosomes in CMs are all of paternal origin; in PMs, they are usually two-thirds paternal, and in normal placentas the proportion is one half.17,18 Therefore, the fact that p57 expression was absent or at low frequency in the tetraploid CMs indicates that all chromosomes in all 30 tetraploid CMs might be paternally derived.

Two tetraploid placentas, with karyotypes of 92,XXYY and 90,XXYY,-13, -14, were diagnosed as HAs and expressed p57. These HAs were considered to be the result of 2 maternal and 2 paternal chromosome sets. The most plausible origin is dispermic (2 haploid sperm) or diplospermic (1 diploid sperm) fertilization of a diploid ovum.6 Diploid ova may result from meiotic failure during oogenesis.19 The remaining 8 HAs (none of which was karyotyped) showed positive p57 staining and were presumably due to 2 sets of chromosomes each from paternal and maternal origin. Analysis of restriction fragment length polymorphisms may show what portions of the chromosomes in tetraploid molar and nonmolar placentas are paternally derived.16,20

In this study, villous or extravillous intermediate trophoblasts stained for p57 in 8 cases of tetraploid CM. This might be due to a relaxation of imprinting during the differentiation from cytotrophoblast to the extravillous trophoblast at the implantation site.21,22 This phenomenon remains to be elucidated.

Expression of p57 is aberrant in tetraploid CMs. This finding is in line with the hypothesis that the loss of p57 is involved in the abnormal development of androgenetic CMs. Although this study did not find any significant differences in p57 expression between persistent and spontaneously resolving cases of tetraploid CM, p57 immunostaining is nonetheless a useful tool for the differential diagnosis of hydatidiform mole. Partial hydatidiform mole and HA usually express p57.8,12,21-24 However, analysis of p57 expression cannot replace histologie analysis. For the treatment of a patient with trophoblastic disease, p57 immunostaining is an ancillary diagnostic method that may be used in concert with flow cytometry.

The author thanks Yukihiro Takeuchi, Yukiko Shiomori, Takashi Honma, and Kuniko Kobayashi for providing technical assistance.

References

1. Kajii T, Ohama K. Androgenetic origin of hydatidiform mole. Nature. 1977; 12:633-634.

2. Szulman AE, Surti U. The syndrome of hydatidiform mole, I: cytogenetic and morphologic correlations. Am J Obstct Cynecol. 1978:131:665-671.

3. Berkowitz RS, Goldstein DP, Bernstein MR. Natural history of partial molar pregnancy. Obstet Gynecol. 1985;66:677-681.

4. Lage JM, Mark SD, Roberts D), Goldstein DP, Bernstein MR, Berkowitz RS. A flow cytometric study of 137 hydropic placentas: correlation between types of hydatidiform moles and nuclear DNA ploidy. Obstet Gynecol. 1992:79:403-410.

5. Fukunaga M, Endo Y, Ushigome S. Clinicopathologic study of tetraploid hydropic villous tissues. Arch Pathol Lab Med. 1996:120:569-572.

6. Lage JM, Weinberg DS, Yavner DL, Bieber FR. The biology of tetraploid hydatidiform moles: histopathology, cytogenetics, and flow cytometry. Hum Pathol. 1989:20:419-423.

7. Matuoka S, Thompson JS, Edwards MC, et al. Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor p57KIP2, on chromosome 11 p15. Proc Natl Acad Sci USA. 1996:93:3026-3030.

8. Chilosi M, Piazzola E, Lestani M, et al. Differential expression of P57KIP2, a maternally imprinted cdk inhibitor, in normal human placenta and gestational trophoblastic disease. Lab Invest. 1998:78:269-276.

9. Chilosi M, Doglioni C, Menestrina F, et al. Expression of extracellular matrix molecules, proliferation markers and cyclin-dependent kinase inhibitors in inflamed tissues. Biochem Soc Trans. 1997;25:524-528.

10. Lee MH, Reynisdottir I, Massague J. Cloning of p57KIP2, a cyclin-dependent kinase inhibitor with unique domain structure and tissue distribution. Genes Dev. 1995:9:639-649.

11. Fukunaga M, Ushigome S, Fukunaga M, Sugishita M. Application of cytometry in diagnosis of hydatidiform moles. Mod Pathol. 1993;6:353-359.

12. Fukunaga M. lmmunohistochemical characterization of p57^sup KIP2^ expression in early hydatidiform moles. Hum Pathol. 2002:33:1188-1192.

13. Medley DW, Freidlander M, Taylor IW, Ruggy CA, Mosgrove EA. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem. 1983:31:1333-1335.

14. Zhang P, Liegeois NJ, Wong C, et al. Altered cell differentiation and proliferation in mice lacking p57KIP2 indicate a role in Beckwith-Wiedemann syndrome. Nature. 1997:387:151-158.

15. Keep D, Zaragoza MV, Hassold T, Redline WR. Very early complete hydatidiform mole. Hum Pathol. 1996:27:708-713.

16. Lawler SD, Fisher RA, Dent J. A prospective genetic study of complete and partial hydatidiform moles. Am J Obstet Cynecol. 1991:164:1270-1277.

17. Surti U, Szulman AE, Wagner K, Leppert MM, O'Brien S. Tetraploid partial moles: two cases with triple paternal contribution and a 92,XXXY karyotype. Hum Genet. 1986:72:15-21.

18. Vejerslev LO, Fisher RA, Surti U, Walke N. Hydatidiform mole: cytogenetically unusual cases and their implications for the present classification. Am J Obstet Gynecol. 1987;157:1 80-1 84.

19. Bieber FR, Moron CC, Nance WE, et al. Genetic studies of an acardiac monster: evidence of polar body twinning in man. Science. 1981:213:775-777.

20. Takahashi M, IkarashiT, Kanazawa K, Kominami R, Tanaka K. Reevaluation of hydatidiform mole by DNA fingerprint method: the discrepancy in the diagnosis by pathological finding and the DNA fingerprint method. Placenta. 1991; 12:487-493.

21. Jun SY, Ro JY, Kim KR. p57^sup kip2^ is useful in the classification and differential diagnosis of complete and partial hydatidiform moles. Histopathology. 2003;43: 17-25.

22. Shih IM, Kurman RJ. The pathology of intermediate trophoblastic tumors and tumor-like lesions. Int J Gynecol Pathol. 2001:20:31-47.

23. Castrillon DH, Sun D, Weremowicz S, Fisher RA, Crum CP, Genest DR. Discrimination of complete hydatidiform mole from its mimics by immunohistochemistry of paternally imprinted gene product p57^sup KIP2^. Am J Surg Pathol. 2001; 25:1225-1230.

24. Crisp H, Burton |L, Stewart R, Wells M. Refining the diagnosis of hydatidiform mole: image ploidy analysis and p57^sup KIP2^ immunohistochemistry. Histopathology. 2003:43:363-373.

Masaharu Fukunaga, MD

Accepted for publication April 20, 2004.

From the Department of Pathology, Jikei Daisan Hospital, Tokyo, Japan.

The author has no relevant financial interest in the products or companies described in this article.

Reprints: Masaharu Fukunaga, MD, Department of Pathology, Jikei Daisan Hospital, 4-11-1, Izumi-honcho, Komae-shi, Tokyo, 201-8601, japan (e-mail: maasafu@jikei.ac.jp).

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