Balkan Journal of Medical Genetics

MOLECULAR AND IMMUNOHISTOCHEMICAL CHARACTERISTICS OF COMPLETE HYDATIDIFORM MOLES
Kubelka-Sabit KB1,*, Prodanova I2, Jasar D1, Bozinovski G3, Filipovski V1, Drakulevski S1, Plaseska-Karanfilska D3
*Corresponding Author: Dr. Katerina B. Kubelka-Sabit, Clinical Hospital Acibadem Sistina, Skupi 5a, 1000 Skopje, Republic of Macedonia. Tel: +389-70-365-338. Fax: +389-23-099-599. E-mail: catkubelka@ yahoo.co.uk; katerina.kubelka@acibademsistina.mk
page: 27

DISCUSSION

The complete hydatidiform mole is mainly diploid and usually has two sets of paternal chromosomes. In 80.0-90.0% of cases, it results from fertilization of an empty egg with a sperm that is then reduplicated in the homozygous diploid genome. In this case, the karyotype is always 46,XX, as 46,YY is non viable. Less often, complete mole is a consequence of dispermic fertilization (diandric diploidy) of an empty egg, so the karyotype can be 46,XY or 46,XX. However, there are rare cases of tetraploid or biparental diploid complete moles [5,8,11-14]. This must be kept in mind when evaluating products of conception, as the diagnosis of complete mole should not be excluded based on molecular analysis alone. It is of great importance not to miss a diagnosis of complete mole, because persistent trophoblastic disease can occur in 15.0-25.0% of these cases [12]. The data in our study correlate with this incidence. In fact, two of the eight patients (25.0%) had persistent trophoblastic disease. One of these patients is still unable to have a successful pregnancy due to recurrent complete moles, whereas the other patient had to have her uterus removed for malignant trophoblastic tumor. The latter patient had twin pregnancy with normal fetus and complete hydatidiform mole. This is a very rare event, occurring in 1 in 20,000 to 1 in 100,000 pregnancies, and can be associated with fetomaternal complications such as persistent gestational trophoblastic disease [15]. The case with twin molar and non molar pregnancy was the only one in our series that was complicated with occurrence of placental site trophoblastic tumor, consistent with the above mentioned results. Other investigators suggest that the risk of persistent trophoblastic disease is the same as for patients with singleton complete molar pregnancy [16]. Although the histopathological criteria for diagnosis of partial and complete molar pregnancy are clearly defined in the literature, unfortunately they are not always present in histological slides, especially in case of early molar pregnancy. For example, in the study of Paul et al. [17], 21 cases of molar pregnancy were reevaluated using flow cytometry and immunohistochemical analysis of p57 marker. It was found that only six of the 21 cases were really molar pregnancies [17]. According to Golfier et al. [4], there is a significant difference in the accuracy of diagnosis of molar preg nancy if the analysis was performed by an experienced pathologist. Apart from the accumulated experience that allows more accurate determination of the histological criteria, this study found that the experienced pathologists significantly more often (41.0%) use additional immunohistochemical markers such as p57 to confirm the histological diagnosis. In fact, these investigators believe that adequate and relevant clinical differentiation between partial, complete molar pregnancy and hydropic abortion can be achieved if the slides are evaluated by an experienced pathologist in this field, but only if additional immunohistochemical or molecular techniques are used [4]. All eight cases in this study were evaluated by an experienced pathologist, with the use of additional immunohistochemical and molecular analyses. In this way, it was possible to confirm the diagnosis of complete mole using immunohistochemistry even in the case of biparental diploidy. Additional immunohistochemical analyses are often used in the literature to distinguish between partial and complete molar pregnancy [2,4,9,10,18]. This method is simple and quick and can be performed without the need for expensive equipment [2]. The most useful antibody to confirm a diagnosis of complete mole is p57 [8,10]. The p57Kip2 protein is a cyclin-dependent kinase inhibitor. It is the protein product of the CDKN1C gene, located on chromosome 11p15.5 and is paternally imprinted and maternally expressed. Thus, since the complete molar pregnancy is missing genetic material from the mother, it is not expected to show CDKN1C gene expression [3,8-10,19]. On the other hand, the cases of hydropic abortions (biparental diploidy) and partial molar pregnancy (diandric triploidy) are expected to show p57 positivity in the cytotrophoblasts and villous stromal cells. However, cases of biparental diploid complete moles also have abnormal, androgenetic pattern of p57Kip2 expression rather than that seen in other types of biparental conceptuses. This is probably due to abnormal imprinting, being a common mechanism underlying the development of biparental diploid complete mole [20]. The immunohistochemical analysis for the p57 marker confirmed all seven cases of androgenetic diploidy as complete molar pregnancies, whereas in the one case of biparental diploidy, the absence of the p57 marker in the cytotrophoblasts and villous stroma was the only back up for the histopathological diagnosis of early complete mole. Other investigators have also been able to differentiate cases of hydropic abortion and partial mole from complete mole cases, based on immunohistochemistry for p57. They have not found p57 positivity in the cytotrophoblasts or villous stroma in any of the complete mole cases tested, unlike partial mole or hydropic abortion [8,10]. The transcription factor p63 is a homologue of the p53 gene and is localized in the long arm of the third chromosome (3q27-29). Its expression in normal placenta is located in the cytotrophoblasts and the protein is considered to have an important role in the maintenance of stem cells, as well as growth and development of various epithelial tissues. Its role in the diagnosis of gestational trophoblastic disease is not yet fully clarified [2,21]. There are conflicting reports regarding the utility of p63 in molar pregnancies. For example, in the study of Erfanian et al. [2], the p63 marker has not shown any positivity in synciciotrophoblasts or cytotrophoblasts in the cases of a non molar pregnancy, while positivity has been found in all cases of partial or complete mole. According to these investigators, p63 may be an ideal marker of differentiation between hydropic abortion and molar pregnancy, but not between partial and complete mole [2]. On the other hand, in the study by Ramalho et al. [21] that included 39 cases of hydropic abortion, molar pregnancy and choriocarcinoma, no statistically significant difference was found in the number of p63-positive cytotrophoblasts between hydropic abortion cases and partial or complete hydatidiform mole. However, they did notice a difference in staining intensity between cases of partial and complete mole [21]. In our opinion, this marker should be tested on a larger series of cases in order to reliably determine its value in the diagnostic algorithm for molar pregnancies. Our one case of recurrent complete hydatidiform mole posed a serious diagnostic difficulty when the molecular results from the second pregnancy were compared to the histopathological results. The morphological changes of the placenta, suggestive of complete mole, were very subtle and pronounced trophoblastic proliferation can sometimes be observed in early normal pregnancies. However, after the patient’s third spontaneous abortion, we insisted on detailed clinical data and found that the patient had previously suffered had at least three early spontaneous abortions, some of them regarded as complete moles based on histopathological findings alone (at different institutions). The histopathological, immunohistochemical and molecular findings from all three products of conception of this patient were re-evalulated and our conclusion was that this was a possible case of familial recurrent hydatidiform mole. These women have an inherited predisposition to diploid biparental molar pregnancies [22]. Most of the cases are due to maternal homozygous and compound heterozygous recessive gene mutations in the NLRP7 gene [23], and less commonly in the KHDC3L gene [7,24]. The NLRP7 gene belongs to the NLRP (nucleotide-binding, leucine-rich repeat, pyrin domain) family and is located at 19q13.4. The NLRP7 protein is thought to be involved in imprinting multiple maternal genes in oocytes that contribute to a developing embryo, ensuring that the genes will be inactive; the corresponding paternal genes are active. This protein is also involved in cell proliferation and differentiation [25]. Its role in inflammation and apoptosis has also been reported [24]. The NLRP7 gene was the first identified recessive gene involved in recurrent hydatidiform mole and mutations have been reported in 48.0-80.0% of patients with more than 50 mutations identified to date [7]. The best chance for our patient to have a normal pregnancy is probably through oocyte donation. Successful pregnancies after recurrent complete moles with oocyte donation have been reported in the literature [26]. It has also been suggested that intracytoplasmic sperm injection, followed by selection of male embryos, can prevent recurrence of complete moles in these patients [27]. Moreover, all three recurrent complete moles in our patient had female genotypes (46,XX). Nonetheless, Fisher et al. [28] published a case of three recurrent biparental moles with two female and one male genotype. Therefore, selection of male embryos is unlikely to prevent complete mole recurrence in these patients [28]. There have been reports of an increased recurrence risk of 0.85-1.0% for women who have experienced an initial complete molar pregnancy in comparison to the normal population [29,30]. However, according to Eagles et al. [29], one in 640 women with complete mole has the rare condition of familial recurrent hydatidiform mole. They had evaluated clinical data from 16,000 women and concluded that recurrent hydatidiform mole accounts for most, but not all, cases of women with three or more complete moles. They have not found significant difference in need for chemotherapy between typical sporadic complete mole and diploid biparental complete mole [29]. Unfortunately, other investigators have also found that neither morphological nor immunohistochemical features can reliably predict subsequent requirements for chemotherapy in complete hydatidiform moles [31]. In our study, there were two cases with persistent hydatidiform mole and need for chemotherapy. One was the case of presumptive familial recurrent hydatidiform mole. The other case of androgenetic diploid complete mole subsequently developed a placental site trophoblastic tumor. Based on our results, although on a limited number of cases, the risk of persistent trophoblastic disease is the same, irrespective of the genetic origin of the complete mole. Different genetic analyses can be used as a complementary method with the classical histopathological analysis. The methods of in situ hybridization and flow cytometry can be used to assess the cell ploidy, in order to distinguish the diploid complete mole or hydropic abortion from the triploid partial mole. However, these analyses cannot distinguish between diploid complete molar pregnancy and diploid hydropic abortion, triploid non molar and partial molar pregnancy, or between mono and dispermic complete mole or androgenetic or biparental complete mole. Molecular genotyping, such as QF-PCR, is a superior compared to the former methods, because it can recognize paternal and maternal alleles [8]. In conclusion, evaluation of the products of conception suspicious for molar pregnancy should be individualized in each laboratory, depending on technical requirements and experience of the staff. If more sophisticated molecular techniques are not available, the pathologist should at least determine the cell ploidy and/or use a p57 immunohistochemical marker. Additional immunohistochemical and molecular methods can considerably aid in the correct diagnosis of a molar pregnancy. Declaration of Interest. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

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