Role of SOX2 in the etiology of embryonal carcinoma, based on analysis of the NCCIT and NT2 cell lines.

The transcription factor SOX2, associated with amongst others OCT3/4, is essential for maintenance of pluripotency and self-renewal of embryonic stem cells. SOX2 is highly expressed in embryonal carcinoma (EC), the stem cell component of malignant nonseminomatous germ cell tumors, referred to as germ cell cancer (GCC). In fact, OCT3/4 together with SOX2 is an informative diagnostic tool for EC in a clinical setting. Several studies support the hypothesis that SOX2 is a relevant oncogenic factor in various cancers and recently, SOX2 has been suggested as a putative therapeutic target for early stage EC. We demonstrate the presence of genomic amplification of SOX2 in an EC cell line, NCCIT, using array comparative genome hybridization and fluorescence in situ hybridization. Down-regulation of SOX2 by targeted siRNA provokes NCCIT cells towards apoptosis, while inhibition of OCT3/4 expression induced differentiation, with retained SOX2 levels. Mice pluripotent xenografts from NCCIT (N-NCCIT and N2-NCCIT) show a consistent SOX2 expression, in spite of loss of the expression of OCT3/4, and differentiation, with retained presence of genomic amplification. No SOX2 amplification has been identified in primary pure and mixed EC in vivo patient samples so far. The data presented in this study are based on a single EC cell line with a SOX2 amplification, with NT2 as control EC cell line, showing no profound induction of apoptosis upon SOX2 downregulation. The findings are of relevance to identify mechanisms involved in the pathogenesis of EC tumors, and support the model of SOX2-oncogene dependency of EC, which however, does not exclude induction of differentiation. This finding is likely related to the presence of wild type p53 in GCC, resulting in expression of downstream target genes, amongst others miR-34a, miR-145 and SOX2, associated to the unique sensitivity of GCC to DNA damaging agents.

Targeted serum miRNA (TSmiR) test for diagnosis and follow-up of (testicular) germ cell cancer patients: a proof of principle.

Germ cell cancers (GCC) are the most frequent malignancy in young Caucasian males. GCC can consist of seminomas (SE) and non-seminomas (malignant NS: embryonal carcinoma (EC), yolk sac tumor (YS), choriocarcinoma (CH) and teratoma (TE)). Current serum-markers used for diagnosis and follow-up (AFP, hCG) are predominantly related to YS and CH and marker positivity can vary during disease. Therefore, stable markers consistently identifying more GCC components, specifically the stem cell components SE and EC, are of interest. Expression of the embryonic stem cell miR-371-3 and miR-302/367 clusters in SE/EC/YS suggest possible application of these micro-RNAs as GCC tumor-markers. The TSmiR protocol constitutes a complete, quality-controlled pipeline for the detection of miRs in serum, based on magnetic bead-based purification and qPCR quantification. As a proof of principle, TSmiR was applied to five independent serum sample series including 80 GCCs, 47 controls, 11 matched pre/post orchidectomy samples and 12 no-GCC testicular masses. GCC serum samples showed a consistent, significant (p < 0.0064) increase of miR-371/372/373/367 levels. Analogous, serum levels returned to baseline after orchidectomy (stage-I disease). Moreover, there was a trend toward higher miR levels in patients with metastasis. These results imply suitability for diagnosis and follow-up. TSmiR showed an overall sensitivity of 98%, clearly outperforming the traditional serum markers AFP/hCG (36%/57%, sensitivity(AFP) = 3%/45%; sensitivity(hCG) = 62%/66%, SE/NS). TSmiR misclassified one tumor as a control. Serum AFP/hCG and TSmiR combined identified all T samples correctly. In conclusion, TSmiR constitutes a highly sensitive and reproducible serum test for GCC patients, suitable to be prospectively tested for diagnostic and follow-up purposes.

Role of stem cell proteins and microRNAs in embryogenesis and germ cell cancer.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst. These cells can proliferate indefinitely and differentiate into all cell lineages. Germ cell cancers (GCC) mimic embryonic development to a certain extent. The origin of GCC trace back to primordial germ cells/gonocytes in the embryo, which determines their specific characteristics such as totipotency and overall (exceptional) sensitivity to DNA damaging agents. Thus GCC provide a useful model system for the study of gene regulation involved in oncogenesis as well as development. Several reports have demonstrated the role of specific proteins and microRNAs (miRs) in the control of pluripotency and thus early development. miRs are small non-coding RNA molecules that post-transcriptionally regulate gene expression by base-paring to protein encoding mRNAs. miRs are predicted to regulate up to 30% of the protein-encoding genes within the human genome. They are expressed in a tissue-specific and developmentally regulated manner. Aberrant miR expression and its correlation with development and progression of cancers is an emerging field. Important evidences have shown that knock-down by synthetic anti-sense oligonucleotides or re-expression of specific miRs by pre-miR can induce drug sensitivity, leading to increased inhibition of cancer cell growth, invasion, and metastasis. In addition, miRs have been found in body fluids of patients with different types of diseases, including cancer. Therefore, investigation of miRs can shed light on the process of pathogenesis, and may provide biomarkers for diagnosis and prognosis. A subset of miRs is specifically expressed in ES cells and GCC, suggesting their critical role in early embryogenesis and development. In this review we discuss the current view of the biology of embryonic stem cell proteins and miRs in GCC, and their potential clinical impact.

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 <1% of most cancers. To provide a more accurate estimate of the frequency of such mutations in TGCTs, we have analysed 96 TGCT samples using high resolution melting curve analysis for sequence variants in these four codons. Although we did not detect any mutations in any of these sites, we did identify a novel mutation (c.1725 R>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.

Prevalence of c-KIT mutations in gonadoblastoma and dysgerminomas of patients with disorders of sex development (DSD) and ovarian dysgerminomas.

Activating c-KIT mutations (exons 11 and 17) are found in 10-40% of testicular seminomas, the majority being missense point mutations (codon 816). Malignant ovarian dysgerminomas represent ~3% of all ovarian cancers in Western countries, resembling testicular seminomas, regarding chromosomal aberrations and c-KIT mutations. DSD patients with specific Y-sequences have an increased risk for Type II Germ Cell Tumor/Cancer, with gonadoblastoma as precursor progressing to dysgerminoma. Here we present analysis of c-KIT exon 8, 9, 11, 13 and 17, and PDGFRA exon 12, 14 and 18 by conventional sequencing together with mutational analysis of c-KIT codon 816 by a sensitive and specific LightCycler melting curve analysis, confirmed by sequencing. The results are combined with data on TSPY and OCT3/4 expression in a series of 16 DSD patients presenting with gonadoblastoma and dysgerminoma and 15 patients presenting pure ovarian dysgerminomas without DSD. c-KIT codon 816 mutations were detected in five out of the total of 31 cases (all found in pure ovarian dysgerminomas). A synonymous SNP (rs 5578615) was detected in two patients, one DSD patient (with bilateral disease) and one patient with dysgerminoma. Next to these, three codon N822K mutations were detected in the group of 15 pure ovarian dysgerminomas. In total activating c-KIT mutations were found in 53% of ovarian dysgerminomas without DSD. In the group of 16 DSD cases a N505I and D820E mutation was found in a single tumor of a patient with gonadoblastoma and dysgerminoma. No PDGFRA mutations were found. Positive OCT3/4 staining was present in all gonadoblastomas and dysgerminomas investigated, TSPY expression was only seen in the gonadoblastoma/dysgerminoma lesions of the 16 DSD patients. This data supports the existence of two distinct but parallel pathways in the development of dysgerminoma, in which mutational status of c-KIT might parallel the presence of TSPY.