Abnormal villous morphology mimicking a hydatidiform mole associated with paternal trisomy of chromosomes 3,7,8 and unipaternal disomy of chromosome 11

The patient was a 36 year-old female, G4P1, having had a normal live birth and two miscarriages. An ultrasound scan at 12 weeks gestation showed no embryo and raised the possibility of hydatidiform mole. Following evacuation of retained products of conception for a missed miscarriage, the patient was referred to the Trophoblastic Tumour and Screening Centre at Charing Cross Hospital with a provisional diagnosis of molar pregnancy. The patient’s serum hCG had reached normal by the time of registration and she remains well with no evidence of persistent trophoblastic neoplasia, delivering a healthy baby boy 29 months after referral to the centre.

Routine haematoxylin and eosin (H&E) stained 4 μm sections were reviewed, using published criteria for distinguishing PHM and CHM from non-molar miscarriage [6‐8], by a specialist trophoblastic disease pathologist with more than 15 years experience in this field. Unstained 4 μm sections were immunostained using a mouse monoclonal primary antibody against p57^KIP2 (Leica Biosystems, Newcastle) at a dilution of 1 in 50, and visualised using the Bond Polymer Refine Detection kit (Leica Biosystems, Newcastle) according to a standard protocol in a CPA approved diagnostic laboratory. Histological review demonstrated villous development consistent with early second trimester but with marked villous dysmorphic features, including variation in size and shape, irregular villous outlines with pseudoinclusions and patchy villous hydropic change closely mimicking hydatidiform mole but morphologically not diagnostic of either PHM or CHM. These changes were variable from field to field, with some more normal appearing villi and other areas with more prominent dysmorphism (Fig. 1). There was limited circumferential trophoblast hyperplasia relative to the degree of villous dysmorphism and no definite karyorrhexis was identified. The morphological differential diagnosis was between a HM and villous dysmorphism secondary to chromosomal abnormality. In view of the unusual morphology in this case, ancillary studies were undertaken including p57^KIP2 immunostaining and molecular genotyping. P57^KIP2 immunostaining showed normal strong nuclear expression in extravillous trophoblast fragments and decidua but with negative staining of villous cytotrophoblast and stroma (Fig. 1).

Initially five areas of placental tissue and a single area of maternal decidua were microdissected from formalin-fixed, paraffin-embedded (FFPE) pathological sections, with reference to a stained H&E section. DNA was then prepared from each sample using a QIAmp DNA FFPE Tissue Kit (Qiagen, Sussex, UK). DNA was subsequently prepared from 500 μl of paternal saliva using an Oragene DNA collection kit (DNA Genotek, Ontario, Canada). 1 μl DNA from the patient, her partner and villous tissue was amplified with primers for 15 short tandem repeat (STR) loci on 13 chromosomes, together with the amelogenin locus, using an AmpFlSTR Identifiler Plus kit (Applied Biosystems, Warrington, UK). PCR products were resolved by capillary electrophoresis using an ABI 3100 Genetic Analyser and genotypes determined using GeneMapper version 4.0 software (Applied Biosystems, Warrington, UK). In order to confirm the results obtained using the AmpFlSTR kit, and to investigate the origin of other chromosomes not represented in the kit, a panel of markers including STRs located on each chromosome was used to amplify DNA from the parental and placental samples, followed by analysis as described above.

Fluorescent microsatellite genotyping of DNA from the parents and villous tissue, using the AmpFlSTR kit, revealed an unusual, but consistent, genotype in all DNA samples from the villous tissue (Table 1). Loci on chromosomes 3, 7, and 8 were trisomic, having three different alleles (D8S1179) or two alleles with one present at twice the dosage of the other (D3S1358; D7S820) (Fig. 2). Loci on chromosome 7 and 8 both had a single maternally derived allele and two alleles from the father while alleles at the D3S1358 locus were not informative with respect to origin. Six informative loci on chromosomes 4, 5, 12, 16 and 18 (Table 1; Fig. 2) exhibited equal parental contributions. Allele sizes for loci on 4 of the remaining chromosomes were consistent with an equal contribution from each parent (Table 1). However, the THO1 locus on chromosome 11 was androgenetic, having a single paternally derived allele (Fig. 2). There was no evidence of mosaicism in the tissue, the genotype of the villi being identical in all five areas of tissue examined. Further amplification with primers for STR loci on chromosomes 3, 7 and 8 confirmed trisomy for both the long and short arms of each chromosome and that the additional chromosome in each case was paternal in origin (Table 2). Amplification with primers for STR loci on the remaining autosomes and the X chromosome were consistent with an equal maternal and paternal contribution to the genome with the exception of chromosome 11. An absence of maternal alleles for six informative loci (Table 2) confirmed that the tissue was androgenetic for chromosome 11. While 4 loci were homozygous for a single paternal allele, the D11S569 and D11S934 loci were heterozygous demonstrating the involvement of two paternal copies of chromosome 11. The villous tissue therefore exhibited paternal disomy for chromosome 3, 7, 8 and 11 together with loss of maternal chromosome 11.

In order to confirm the observations resulting from genotyping, fluorescence in situ hybridization (FISH) was performed on sections of the villous tissue. FFPE sections of the placental tissue were pretreated and hybridized using the ThermoBrite Elite according to manufacturer’s protocols (Leica Biosystems, Nussloch, Germany). FISH was then performed using a panel of DNA probes for chromosomes 3 (MECOM; 3q26), 7 (7q22 & 7q36), 8 (MYC; 8q24), 11 (KMT2A; 11q23), 19 (ERCC1; 19q13 & ZNF443; 19p13) and the X chromosome (DXZ1); all probes were from Kreatech (Leica Biosystems). Sections were analysed and focus stacked images were captured using the Isis imaging system (MetaSystems, Altlussheim, Germany), again, with reference to a stained H&E section. Hybridization with probes for chromosome 3, 7 and 8 confirmed trisomy for each of these chromosomes. Probes for the X chromosome, chromosome 19 and chromosome 11 supported the observation that other chromosomes were disomic (Fig. 3).