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

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

DNA methylation profiles delineate epigenetic heterogeneity in seminoma and non-seminoma

Background: It remains important to understand the biology and identify biomarkers for less studied cancers like testicular cancer. The purpose of this study was to determine the methylation frequency of several cancer-related genes in different histological types of testicular cancer and normal testis tissues (NT). Methods: DNA was isolated from 43 seminomas (SEs), 14 non-SEs (NSEs) and 23 NT, and was assayed for promoter methylation status of 15 genes by quantitative methylation-specific PCR. The methylation status was evaluated for an association with cancer, and between SEs and NSEs. Results: We found differential methylation pattern in SEs and NSEs. MGMT, VGF, ER-Β and FKBP4 were predominately methylated in NSEs compared with SEs. APC and hMLH1 are shown to be significantly more methylated in both subtypes in comparison with NT. When combining APC, hMLH1, ER-Β and FKBP4, it is possible to identify 86% of the NSEs, whereas only 7% of the SEs. Conclusions: Our results indicate that the methylation profile of cancer-associated genes in testicular cancer correlates with histological types and show cancer-specific pattern for certain genes. Further methylation analysis, in a larger cohort is needed to elucidate their role in testicular cancer development and potential for therapy, early detection and disease monitoring

Genomic copy number and expression patterns in testicular germ cell tumours

Testicular germ cell tumours of adults and adolescents (TGCT) include seminomas (SE) and nonseminomas (NS), with spermatocytic seminomas (SSE) representing a distinct entity in older men. SE and NS have gain of 12p material in all cases, whereas SSE are associated with overrepresentation of chromosome 9. Here, we compare at the chromosomal level, copy number imbalances with global expression changes, identified by comparative expressed sequence hybridisation analyses, in seven SE, one combined tumour, seven NS and seven cell lines. Positive correlations were found consistent with copy number as a main driver of expression change, despite reported differences in methylation status in SE and NS. Analysis of chromosomal copy number and expression data could not distinguish between SE and NS, in-keeping with a similar genetic pathogenesis. However, increased expression from 4q22, 5q23.2 and 9p21 distinguished SSE from SE and NS and decreased copy number and expression from 2q36–q37 and 6q24 was a specific feature of NS-derived cell lines. Our analysis also highlights 19 regions with both copy number and expression imbalances in greater than 40% of cases. Mining available expression array data identified genes from these regions as candidates for involvement in TGCT development. Supplementary data is available at http://www.crukdmf.icr.ac.uk/array/array.html

Malignant germ cell tumours of childhood: new associations of genomic imbalance

Malignant germ cell tumours (MGCTs) of childhood are a rare group of neoplasms that comprise many histological subtypes and arise at numerous different sites. Genomic imbalances have been described in these tumours but, largely because of the paucity of cases reported in the literature, it is unclear how they relate to abnormalities in adult MGCTs and impact on potential systems for classifying GCTs. We have used metaphase-based comparative genomic hybridisation to analyse the largest series of paediatric MGCTs reported to date, representing 34 primary tumours (22 yolk sac tumours (YSTs), 11 germinomatous tumours and one metastatic embryonal carcinoma) occurring in children from birth to age 16, including 17 ovarian MGCTs. The large dataset enabled us to undertake statistical analysis, with the aim of identifying associations worthy of further investigation between patterns of genomic imbalance and clinicopathological parameters. The YSTs showed an increased frequency of 1p- (P=0.003), 3p+ (P=0.02), 4q− (P=0.07) and 6q− (P=0.004) compared to germinomatous tumours. Gain of 12p, which is invariably seen in adult MGCTs, was present in 53% of primary MGCTs of children aged 5–16 and was also observed in four of 14 YSTs affecting children less than 5. Two of these cases (14% of MGCTs in children less than 5) showed gain of the 12p11 locus considered to be particularly relevant in adult MGCTs. Gain of 12p showed a significant association with gain of 12q. Conversely, MGCTs without 12p gain displayed a significantly increased frequency of loss on 16p (P=0.04), suggesting that this imbalance may contribute to tumour development in such cases. This data provides new insight into the biology of this under-investigated tumour group and will direct future studies on the significance of specific genetic abnormalities

The INeS study: prevention of multiple pregnancies: a randomised controlled trial comparing IUI COH versus IVF e SET versus MNC IVF in couples with unexplained or mild male subfertility

BACKGROUND Multiple pregnancies are high risk pregnancies with higher chances of maternal and neonatal mortality and morbidity. In the past decades the number of multiple pregnancies has increased. This trend is partly due to the fact that women start family planning at an increased age, but also due to the increased use of ART. Couples with unexplained or mild male subfertility generally receive intrauterine insemination IUI with controlled hormonal stimulation (IUI COH). The cumulative pregnancy rate is 40%, with a 10% multiple pregnancy rate. This study aims to reveal whether alternative treatments such as IVF elective Single Embryo Transfer (IVF e SET) or Modified Natural Cycle IVF (MNC IVF) can reduce the number of multiple pregnancy rates, but uphold similar pregnancy rates as IUI COH in couples with mild male or unexplained subfertility. Secondly, the aim is to perform a cost effective analyses and assess treatment preference of these couples. METHODS/DESIGN We plan a multicentre randomised controlled clinical trial in the Netherlands comparing six cycles of intra-uterine insemination with controlled ovarian hyperstimulation or six cycles of Modified Natural Cycle (MNC) IVF or three cycles with IVF-elective Single Embryo Transfer (eSET) plus cryo-cycles within a time frame of 12 months. Couples with unexplained subfertility or mild male subfertility and a poor prognosis for treatment independent pregnancy will be included. Women with anovulatory cycles, severe endometriosis, double sided tubal pathology or serious endocrine illness will be excluded. Our primary outcome is the birth of a healthy singleton. Secondary outcomes are multiple pregnancy, treatment costs, and patient experiences in each treatment arm. The analysis will be performed according tot the intention to treat principle. We will test for non-inferiority of the three arms with respect to live birth. As we accept a 12.5% loss in pregnancy rate in one of the two IVF arms to prevent multiple pregnancies, we need 200 couples per arm (600 couples in total). DISCUSSION Determining the safest and most cost-effective treatment will ensure optimal chances of pregnancy for subfertile couples with substantially diminished perinatal and maternal complications. Should patients find the most cost-effective treatment acceptable or even preferable, this could imply the need for a world wide shift in the primary treatment. TRIAL REGISTRATION Current Controlled Trials ISRCTN 52843371Alexandra J Bensdorp, Els Slappendel, Carolien Koks, Jur Oosterhuis, Annemieke Hoek, Peter Hompes, Frank Broekmans, Harold Verhoeve, Jan Peter de Bruin, Janne Meije van Weert, Maaike Traas, Jacques Maas, Nicole Beckers, Sjoerd Repping, Ben W Mol, Fulco van der Veen and Madelon van Wel