Heterogeneous X inactivation in trophoblastic cells of human full-term female placentas

In female mammalian cells, one of the two X chromosomes is inactivated to compensate for gene-dose effects, which would be otherwise doubled compared with that in male cells. In somatic lineages in mice, the inactive X chromosome can be of either paternal or maternal origin, whereas the paternal X chromosome is specifically inactivated in placental tissue. In human somatic cells, X inactivation is mainly random, but both random and preferential paternal X inactivation have been reported in placental tissue. To shed more light on this issue, we used PCR to study the methylation status of the polymorphic androgen-receptor gene in full-term human female placentas. The sites investigated are specifically methylated on the inactive X chromosome. No methylation was found in microdissected stromal tissue, whether from placenta or umbilical cord. Of nine placentas for which two closely apposed samples were studied, X inactivation was preferentially maternal in three, was preferentially paternal in one, and was heterogeneous in the remaining five. Detailed investigation of two additional placentas demonstrated regions with balanced (1:1 ratio) preferentially maternal and preferentially paternal X inactivation. No differences in ratio were observed in samples microdissected to separate trophoblast and stromal tissues. We conclude that methylation of the androgen receptor in human full-term placenta is specific for trophoblastic cells and that the X chromosome can be of either paternal or maternal origin

DICER1 RNase IIIb domain mutations are infrequent in testicular germ cell tumours

Background: Testicular Germ Cell Tumours (TGCT) are the most frequently occurring malignancy in males from 15-45 years of age. They are derived from germ cells unable to undergo physiological maturation, although the genetic basis for this is poorly understood. A recent report showed that mutations in the RNase IIIb domain of DICER1, a micro-RNA (miRNA) processing enzyme, are common in non-epithelial ovarian cancers. DICER1 mutations were found in 60% of Sertoli-Leydig cell tumours, clustering in four codons encoding metal-binding sites. Additional analysis of 14 TGCT DNA samples identified one case that also contained a mutation at one of these sites. Findings. A number of previous studies have shown that DICER1 mutations are found in Q) within the RNase IIIb domain in one TGCT sample, which was predicted to disturb DICER1 function. Conclusion: Overall our findings suggest a mutation frequency in TGCTs of ∼1%. We conclude therefore that hot-spot mutations, frequently seen in Sertoli-Leydig cell tumours, are not common in TGCTs

Cripto: Expression, epigenetic regulation and potential diagnostic use in testicular germ cell tumors

Type II germ cell tumors arise after puberty from a germ cell that was incorrectly programmed during fetal life. Failure of testicular germ cells to properly differentiate can lead to the formation of germ cell neoplasia in situ of the testis; this precursor cell invariably gives rise to germ cell cancer after puberty. The Nodal co-receptor Cripto is expressed transiently during normal germ cell development and is ectopically expressed in non-seminomas that arise from germ cell neoplasia in situ, suggesting that its aberrant expression may underlie germ cell dysregulation and hence germ cell cancer. Here we investigated methylation of the Cripto promoter in mouse germ cells and human germ cell cancer and correlated this with the level of CRIPTO protein expression. We found hypomethylation of the CRIPTO promoter in undifferentiated fetal germ cells, embryonal carcinoma and seminomas, but hypermethylation in differentiated fetal germ cells and the differentiated types of non-seminomas. CRIPTO protein was strongly expressed in germ cell neoplasia in situ along with embryonal carcinoma, yolk sac tumor and seminomas. Further, cleaved CRIPTO was detected in media from seminoma and embryonal carcinoma cell lines, suggesting that cleaved CRIPTO may provide diagnostic indication of germ cell cancer. Accordingly, CRIPTO was detectable in serum from 6/15 patients with embryonal carcinoma, 5/15 patients with seminoma, 4/5 patients with germ cell neoplasia in situ cells only and in 1/15 control patients. These findings suggest that CRIPTO expression may be a useful serological marker for diagnostic and/or prognostic purposes during germ cell cancer management

Human Germ Cell Tumors are Developmental Cancers: Impact of Epigenetics on Pathobiology and Clinic

Current (high throughput omics-based) data support the model that human (malignant) germ cell tumors are not initiated by somatic mutations, but, instead through a defined locked epigenetic status, representative of their cell of origin. This elegantly explains the role of both genetic susceptibility as well as environmental factors in the pathogenesis, referred to as ‘genvironment’. Moreover, it could also explain various epidemiological findings, including the rising incidence of this type of cancer in Western societies. In addition, it allows for identification of clinically relevant and informative biomarkers both for diagnosis and follow-up of individual patients. The current status of these findings will be discussed, including the use of high throughput DNA methylation profiling for determination of differentially methylated regions (DMRs) as well as chromosomal copy number variation (CNV). Finally, the potential value of methylation-specific tumor DNA fragments (i.e., XIST promotor) as well as embryonic microRNAs as molecular biomarkers for cancer detection in liquid biopsies will be presented

Human Germ Cell Tumors are Developmental Cancers: Impact of Epigenetics on Pathobiology and Clinic

Current (high throughput omics-based) data support the model that human (malignant) germ cell tumors are not initiated by somatic mutations, but, instead through a defined locked epigenetic status, representative of their cell of origin. This elegantly explains the role of both genetic susceptibility as well as environmental factors in the pathogenesis, referred to as ‘genvironment’. Moreover, it could also explain various epidemiological findings, including the rising incidence of this type of cancer in Western societies. In addition, it allows for identification of clinically relevant and informative biomarkers both for diagnosis and follow-up of individual patients. The current status of these findings will be discussed, including the use of high throughput DNA methylation profiling for determination of differentially methylated regions (DMRs) as well as chromosomal copy number variation (CNV). Finally, the potential value of methylation-specific tumor DN

Specific detection of OCT3/4 isoform A/B/B1 expression in solid (germ cell) tumours and cell lines: confirmation of OCT3/4 specificity for germ cell tumours

BACKGROUND: OCT3/4 (POU5F1) is an established diagnostic immunohistochemical marker for specific histological variants of human malignant germ cell tumours (GCTs), including the seminomatous types and the stem cell component of non-seminomas, known as embryonal carcinoma. OCT3/4 is crucial for the regulation of pluripotency and the self-renewal of normal embryonic stem-and germ cells. Detection of expression of this transcription factor is complicated by the existence of multiple pseudogenes and isoforms. Various claims have been made about OCT3/4 expression in non-GCTs, possibly related to using nonspecific detection methods. False-positive findings undermine the applicability of OCT3/4 as a specific diagnostic tool in a clinical setting. In addition, false-positive findings could result in misinterpretation of pluripotency regulation in solid somatic cancers and their stem cells. Of the three identified isoforms - OCT4A, OCT4B and OCT4B1 - only OCT4A proved to regulate pluripotency. Up until now, no convincing nuclear OCT4A protein expression has been shown in somatic cancers or tissues. METHODS: This study investigates expression of the various OCT3/4 isoforms in GCTs (both differentiated and undifferentiated) and somatic (non-germ cell) cancers, including representative cell lines and xenografts. RESULTS: Using specific methods, OCT4A and OCT4B1 are shown to be preferentially expressed in undifferentiated GCTs. The OCT4B variant shows no difference in expression between GCTs (either differentiated or undifferentiated) and somatic cancers. In spite of the presence of OCT4A mRNA in somatic cancer-derived cell lines, no OCT3/4 protein is detected. Significant positive correlations between all isoforms of OCT3/4 were identified in both tumours with and without a known stem cell component, possibly indicating synergistic roles of these isoforms. CONCLUSION: This study confirms that OCT4A protein only appears in seminomatous GCTs, embryonal carcinoma and representative cell lines. Furthermore, it emphasises that in order to correctly assess the presence of functional OCT3/4, both isoform specific mRNA and protein detection are required. British Journal of Cancer (2011) 105, 854-863. doi: 10.1038/bjc.2011.270 www.bjcancer.com Published online 16 August 2011 (C) 2011 Cancer Research U

Serum and CSF microRNAs in paediatric malignant GCTs

BACKGROUND: The current biomarkers alpha-fetoprotein and human chorionic gonadotropin have limited sensitivity and specificity for diagnosing malignant germ-cell tumours (GCTs). MicroRNAs (miRNAs) from the miR-371-373 and miR-302/367 clusters are overexpressed in all malignant GCTs, and some of these miRNAs show elevated serum levels at diagnosis. Here, we developed a robust technical pipeline to quantify these miRNAs in the serum and cerebrospinal fluid (CSF). The pipeline was used in samples from a cohort of exclusively paediatric patients with gonadal and extragonadal malignant GCTs, compared with appropriate tumour and non-tumour control groups. METHODS: We developed a method for miRNA quantification that enabled sample adequacy assessment and reliable data normalisation. We performed qRT-PCR profiling for miR-371-373 and miR-302/367 cluster miRNAs in a total of 45 serum and CSF samples, obtained from 25 paediatric patients. RESULTS: The exogenous non-human spike-in cel-miR-39-3p and the endogenous housekeeper miR-30b-5p were optimal for obtaining robust serum and CSF qRT-PCR quantification. A four-serum miRNA panel (miR-371a-3p, miR-372-3p, miR-373-3p and miR-367-3p): (i) showed high sensitivity/specificity for diagnosing paediatric extracranial malignant GCT; (ii) allowed early detection of relapse of a testicular mixed malignant GCT; and (iii) distinguished intracranial malignant GCT from intracranial non-GCT tumours at diagnosis, using CSF and serum samples. CONCLUSIONS: The pipeline we have developed is robust, scalable and transferable. It potentially promises to improve clinical management of paediatric (and adult) malignant GCTs.Grant funding was from CwCUK/GOSHCC (M.J. Murray, K.L. Raby, J.C. Nicholson, N. Coleman), SPARKS (M.J. Murray, J.C. Nicholson, N. Coleman), AstraZeneca (E. Bell, H. Brown and B. Destenaves), CRUK (N. Coleman), MRC (M.J. Murray) and an Erasmus MC MRACE grant (M.A. Rijlaarsdam). The authors also thank the Max Williamson Fund and The Perse Preparatory School, Cambridge for supporting this study.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/bjc.2015.42