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

MATERIALS AND METHODS

A total of 367 consecutive cases of spontaneous abortion were analyzed in a 3-year period. Apart from standard microscopic analysis, additional molecular and immunohistochemical analyses were performed in all cases suspicious for complete molar pregnancy. A diagnosis of complete molar pregnancy was suspected in eight cases, based on the following histomorphological criteria: 1) presence of a heterogeneous population of chorionic villi, ranging from small to extremely large villi with pronounced edema and frequent finding of central cavitations; 2) mucous stromal degeneration; 3) karyorrhectic debris in the stroma, and 4) proliferation of the sinciciotrophoblasts and cytotro-phoblasts [6] (Figures 1 and 2). For three of the cases, fragments of placental and decidual tissue were separated prior to fixation in formalin, during the macroscopic analysis of the products of conception. This process was done by an experienced pathologist. One set of placental and decidual tissue, containing at least ~30 mm3 of tissue in each container, was frozen in liquid nitrogen. A separate set of placental and decidual tissue fragments were fixed in formalin, routinely processed and embedded in paraffin, for routine histopathological and immunohistochemical analysis. For the remaining five cases, a small ~30 mm3 fragment of formalin-fixed and paraffin-embedded placental and decidual tissue was taken from the processed paraffin blocks. The decidual tissue was used for extraction of maternal DNA (deoxyribonucleic acid), in cases where a maternal blood sample could not be obtained. For the immunohistochemical analysis, primary mouse monoclonal antibodies p57 (clone 25B2) from Vector Laboratories (Burlingame, CA, USA) and Ki-67 and p63 (clone 4A4) from Ventana (Tucson, AZ, USA) were used. For the p57 antibody detection, EnVision FLEX+ kit from Dako from Agilend Technologies (Santa Clara, CA, USA) was used. The reaction was performed on an automated platform for immunohistochemistry Autostainer Link 48 from Dako from Agilent Technologies. For the p63 and Ki-67 antibody detection, ultraView Universal DAB Detection Kit from Ventana was used on an automated platform Ventana BenchMark GX. The type of the antibodies, clones and dilutions are given in Table 1. For all antibodies used, a reaction was considered as positive if a brown signal was observed in the cell nuclei. The positivity for the antibodies was evaluated in the cytotrophoblastic cells as percentage of positive cells. The staining intensity for p57 and p63 antibodies was evaluated as: 0 (no staining); [+] (weak staining); [++] (moderate staining); [+++] (intense staining). In order to evaluate the ploidy and to distinguish maternal from paternal allelles, a molecular analysis of placental and decidual tissue (or maternal blood cells) was performed. The quantitative fluorescent polymerase chain reaction (QF-PCR) molecular method was performed on ABI-PRISM 3100 and 3500 Genetic Analyzers (Applied Biosystems, Foster City, CA, USA). In a multiplex PCR reaction, four DNA markers on chromosomes 18 (D18 S535, D18S391, D18S390 and D18S386), 21 (D21S1435, D21S1446, D21S1411 and D21S1414), 13 (D13S631, D13 S305, D13S258 and D13S1817), two markers on the X chromosome (DXS6803 and HPRT), and amelogenin locus (AMXS) were analyzed.



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