A total of 51 GTNs were histologically reviewed, including 19 choriocarcinomas, 17 ETTs, and 15 PSTTs (). Of these samples, ETT17 contained a small area of choriocarcinoma and PSTT15 contained a focal ETT component. Representative histologic features of the GTNs are illustrated in . All cases yielded informative results in at least one of the gene markers utilized for the sex chromosome genotyping. We found that all informative cases contained a X-chromosomal complement while only five (10%, 95% CI: 18.2%–1.8%) of 51 tumors harbored a Y chromosome complement (). Specifically, Y-chromosomal signals were detected in one (5%) of 19 choriocarcinomas, one (7%) of 15 PSTTs, and three (18%) of 17 ETTs (). illustrates the genotypes in representative specimens. Of note, the genotypes were identical in genomic DNA obtained from different tissue blocks of the same case. For those specimens with a Y-chromosome, all three gene markers revealed consistent outcomes, although the relative abundance of a Y gene versus an X gene varied. For example, PSTT5 showed a small Y peak in both amelogenin and ZF loci that could make the Y assignment equivocal (). However, by analyzing PRKY, we clearly detected a robust PRKY peak from the same specimen. Similarly, ETT12 contained a relatively small amelogenin Y peak but had significantly large peaks at both PRKY and ZFY. These findings indicate the variable efficiency of primers that amplify the different Y loci of the three genes on formalin-fixed paraffin tissues and underscore the importance to include additional markers to assess the presence of Y-chromosomal elements. The histopathological features in those tumors with a Y-chromosome were indistinguishable from those without a Y-chromosome. The percentage of cases showing Y peaks is listed in .
The sex chromosome assignment in all the GTN samples.
Figure 1 Histological features of gestational trophoblastic neoplasms. Choriocarcinoma is characterized by biphasic growth pattern composed of syncytiotrophoblast and mononucleate trophoblastic cells, forming vasculogenic mimicry. Placental site trophoblastic (more ...)
Figure 2 Genotypes in representative trophoblastic tumor specimens. The presence of either the X or Y-chromosome in GTNs was determined by the analysis of three genes that have X and Y homologues distinguishable by their PCR product size; Amelogenin X and Y ( (more ...)
Summary of percentage of tumor cases positive for a Y allele for at least one marker.
The lack of a Y-chromosomal complement in the majority of GTNs is intriguing and several theories can account for this phenomenon. The most likely cause of the phenomenon is that Y-chromosomal deletions have no functional effects on tumor progression [15
]. In this case, the absence of Y chromosome in GTNs may simply reflect the fact that many GTNs develop from complete hydatidiform moles of which approximately 90% contain a karyotype of 46,XX due to fertilization of an “empty” ovum (without nucleus) by a single haploid (23X) sperm followed by haploid genome duplication [10
]. Thus, the GTNs that develop from complete hydatidiform moles retain the same sex chromosome assignment as their precursors and do not harbor a Y-chromosome. While 90% of complete hydatidiform moles arise from monospermy, approximately 10% are due to fertilization of an empty ovum with two sperm. Half of these cases that arise from dispermy would be expected to carry a Y-chromosome. Thus it could be predicted that approximately 5% of complete hydatidiform moles, and their resulting choriocarcinomas, would carry a Y-chromosome, which is exactly the percentage we obtained in this study.
Although the above represents our favorite view, other interpretations should also be indicated. It is possible that Y-chromosome deletions have a functional implication in the development of GTNs. In addition to GTNs developing from trophoblastic cells of a female conceptus, it can be speculated that GTNs arising from trophoblastic cells of a male conceptus will undergo clonal selection of trophoblastic cells with a deleted Y-chromosome due to their underlying genomic instability. In both scenarios, it is assumed that the presence of a Y-chromosome is not compatible with tumor initiation, possibly due to potential growth-inhibitory effects conferred by the products of genes located in the Y-chromosome. In support of this notion is the observation of a small but unambiguous Y peak of AMELY
in the carefully dissected ETT17 (). Also, previous reports have demonstrated Y-chromosome loss in several types of human cancer including prostate carcinoma, renal cell carcinoma, acute promyelocytic leukemia, and head and neck squamous carcinoma [15
Lastly, the lack of Y-chromosome detection in other studies may be the result of micro-deletions in the Y-chromosomal regions analyzed, yielding a false negative result. This technical pitfall has been well documented in solid tumors when the amelogenin-based assay was applied [13
]. To overcome this problem, in this study we have included two additional gene markers, PRK
, along with the standard amelogenin test. Similar to the Amelogenin (AMEL)
locus, the PRK,
genes have X and Y homologues located on Xp (PRKX
) and Yp (ZFX
). The PRKY
are located 3.9
Mb telomeric to AMELY
and 0.35 Mb centromeric to AMELY,
respectively. The failure to detect any of the three genes of the Y chromosome derives a more definitive conclusion and suggests that the absence of Y-chromosome is not likely due to somatic micro-deletions or microsatellite instability of the Y-chromosome-associated loci in GTNs.
Among 51 GTNs analyzed, we detected Y alleles in five tumors based on the presence of Y peaks in at least one of the AMELY
loci. Among these five tumors was a PSTT. This finding is in contrast to a previous report demonstrating that none of 13 PSTTs harbored the AMELY
]. The discrepancy is likely explained by the larger sample size and the additional Y markers employed in this study. The conclusion from the current study is also different from our previous report showing that approximately half of PSTTs and ETTs contained the sex-determining region Y (SRY) on Y chromosome [19
]. In that study, a high cycle number of PCR amplification was used in order to detect a limited source of genomic DNA from paraffin tissues, raising the possibility of nonspecific amplification from contaminants. Thus, we believe that the results from the current study are more definitive in determining the sex chromosome assignment of GTNs.
In conclusion, this study provides a comprehensive analysis of sex chromosome distributions in all types of GTNs using three independent gene markers with differing PCR product lengths in the X and Y-chromosomes when specific primer pairs are used. Our results, based on a relatively large number of cases, clearly demonstrate the presence of a distinct but low Y-chromosomal complement in choriocarcinomas, PSTTs, and ETTs, that contributes to an overall figure of approximately 10%. It is most likely that the shortfall of Y chromosomal complements in GTNs may simply be due to the genetic basis of their precursor lesions, complete hydatidiform moles in which the majority of cases had the genotype of XX [20
]. In conclusion, our results suggest that the majority of GTNs are preceded by antecedent complete molar pregnancy, many of which may be under recognized as the early complete moles usually lack the characteristic histopathological features.