J. W. Oosterhuis

Differential expression of SOX17 and SOX2 in germ cells and stem cells has biological and clinical implications

Combined action of SOX and POU families of transcription factors plays major roles in embryonic development. In embryonic stem cells, the combination of SOX2 and POU5F1 (OCT3/4) is essential for maintaining the undifferentiated state by activating pluripotency‐linked genes, and inhibition of genes involved in differentiation. Besides embryonic stem cells, POU5F1 is also present in early germ cells, primordial germ cells, and gonocytes, where it has a role in suppression of apoptosis. Here we demonstrate that SOX2 is absent in germ cells of human fetal gonads, and as expected carcinoma in situ (CIS), ie the precursor lesion of testicular germ cell tumours of adolescents and adults (TGCTs), and seminoma. Based on genome‐wide expression profiling, SOX17 was found to be present, instead of SOX2, in early germ cells and their malignant counterparts, CIS and seminoma. Immunohistochemistry, western blot analysis, and quantitative RT‐PCR showed that SOX17 is a suitable marker to distinguish seminoma from embryonal carcinoma, confirmed in representative cell lines. Aberrant SOX2 expression can be present in Sertoli cells when associated with CIS, which can be misdiagnosed as embryonal carcinoma. In conclusion, this study demonstrates the absence of SOX2 in human embryonic and malignant germ cells, which express SOX17 in conjunction with POU5F1. This finding has both diagnostic and developmental biological implications. It allows the identification of seminoma‐like cells from embryonal carcinoma based on a positive marker and might be the explanation for the different function of POU5F1 in normal and malignant germ cells versus embryonic stem cells. Copyright

Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11.2–p12.1

Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2–p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2–p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2–p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2–p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2–p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development.

Expression and interdependencies of pluripotency factors LIN28, OCT3/4, NANOG and SOX2 in human testicular germ cells and tumours of the testis

OCT3/4, NANOG, SOX2 and, most recently, LIN28 have been identified as key regulators of pluripotency in mammalian embryonic and induced stem cells, and are proven to be crucial for generation of the mouse germ-cell lineage. These factors are a hallmark of certain histological types of germ-cell tumours (GCTs). Here, we report novel information on the temporal and spatial expression pattern of LIN28 during normal human male germ-cell development as well as various types of GCTs. To investigate LIN28 expression, immunohistochemical analyses and quantitative proximity ligation assay-based TaqMan protein assays were applied on snap-frozen and formalin-fixed, paraffin-embedded samples as well as representative cell lines. LIN28 was found in primordial germ cells, gonocytes and pre-spermatogonia, in contrast to OCT3/4 and NANOG, which were found only in the first two stages. LIN28 was also found in all precursor lesions (carcinoma in situ and gonadoblastoma) of type II GCTs, as well as the invasive components seminoma and the non-seminomatous elements embryonal carcinoma and yolk sac tumour. Choriocarcinoma showed a heterogeneous pattern, while teratomas and spermatocytic seminomas (type III GCTs) were negative. This expression pattern suggests that LIN28 is associated with malignant behaviour of type II GCTs. Cell line experiments involving siRNA knockdown of LIN28, OCT3/4 and SOX2 showed that LIN28 plays a role in the maintenance of the undifferentiated state of both seminoma and embryonal carcinoma, closely linked to, and likely upstream of OCT3/4 and NANOG. In conclusion, LIN28 regulates the differentiation status of seminoma and embryonal carcinoma and is likely to play a related role in normal human germ-cell development.

FOXL2 and SOX9 as parameters of female and male gonadal differentiation in patients with various forms of disorders of sex development (DSD)

The transcription factors SOX9 and FOXL2 are required for male and female mammalian gonadal development. We have used specific antibodies to investigate the role of these key proteins in disorders of sex development (DSD), specifically inter‐sex states. In normal gonads, SOX9 was found to be restricted to the presence of (pre‐)Sertoli cells, while FOXL2 was found in granulosa cells, and in stromal cells interpreted as early ovarian stroma. Both proteins were found within a single patient, when testicular and ovarian development was present; and within the same gonad, when both differentiation lineages were identified, as in ovotesticular DSD (ie hermaphrodite). Especially SOX9 was informative to support the presence of early testicular development (ie seminiferous tubules), expected based on morphological criteria only. In a limited number of DSD cases, FOXL2 was found within reasonably well‐developed seminiferous tubules, but double staining demonstrated that it was never strongly co‐expressed with SOX9 in the same cell. All seminiferous tubules containing carcinoma in situ (CIS), the malignant counterpart of a primordial germ cell, ie the precursor of type II germ cell tumours of the testis, seminomas and non‐seminomas, showed the presence of SOX9 and not FOXL2. In contrast, gonadoblastomas (GBs), the precursor of the same type of cancer, in a dysgenetic gonad, showed expression of FOXL2 and no, or only very low, SOX9 expression. These findings indicate that gonadal differentiation, ie testicular or ovarian, determines the morphology of the precursor of type II germ cell tumours, CIS or GB, respectively. We show that in DSD patients, the formation of either ovarian or/and testicular development can be visualized using FOXL2 and SOX9 expression, respectively. In addition, it initiates a novel way to study the role of the supportive cells in the development of either CIS or GB. Copyright

Dissecting the molecular pathways of (testicular) germ cell tumour pathogenesis; from initiation to treatment-resistance.

Human type II germ cell tumours (GCTs) originate from an embryonic germ cell, either as a primordial germ cell or gonocyte. This start determines the biological as well as clinical characteristics of this type of cancer, amongst others their totipotency as well as their overall (exceptional) sensitivity to DNA damaging agents. The histology of the precursor lesion, either carcinoma in situ or gonadoblastoma, depends on the level of testicularization (i.e. testis formation) of the gonad. The impact of either intrinsic (genetic) - and environmental factors involved in the pathogenesis is demonstrated by disorders of sex development as well as testicular dysgenesis syndrome as risk factors, including cryptorchidism, hypospadias and disturbed fertility as parameters. This knowledge allows identification of individuals at risk for development of this type of cancer, being a population of interest for screening. Factors known to regulate pluripotency during embryogenesis are proven to be of diagnostic value for type II GCTs, including OCT3/4, even applicable for non-invasive screening. In addition, presence of stem cell factor, also known as KITLG, allows distinction between delayed matured germ cells and the earliest stages of malignant transformation. This is of special interest because of the identified association between development of type II GCTs of the testis and a limited number of single nucleotide polymorphisms, including some likely related to KITL. Transition from the precursor lesion to an invasive cancer is associated with gain of the short arm of chromosome 12, in which multiple genes might be involved, including KRAS2 and possibly NANOG (pseudogenes). While most precursor lesions will progress to an invasive cancer, only a limited number of cancers will develop treatment resistance. Putative explanatory mechanisms are identified, including presence of microsatellite instability, BRAF mutations, apoptosis suppression and p21 sub-cellular localization. It remains to be investigated how these different pathways integrate to each other and how informative they are at the patient-individual level. Further understanding will allow development of more targeted treatment, which will benefit quality of life of these young cancer patients.

Gonadal and extragonadal germ cell tumours in the United States, 1973-2007

Germ cell tumours (GCTs) most often arise in the gonads, but some develop extragonadally. The aim of this study was to examine gender- and race-specific trends in incidence and survival of gonadal (GGCTs) and extragonadal GCTs (EGCTs) in the US from 1973 to 2007. We also examined the topographical distribution of EGCTs by race and gender. We estimated age-specific and age-standardized incidence rates and 5-year relative survival rates (RSR) of GCTs using the Surveillance, Epidemiology and End Results (SEER) Program (SEER nine registries). GCTs and their topographical sites were identified using ICD-O morphology and topography codes. Of 21,170 GCTs among males, 5.7% were extragonadal (Whites 5.5%; Blacks 16.3%). Of 2093 GCTs among females, 39.3% were extragonadal (Whites, 36.9%; Blacks 51.0%). The incidence of GGCT was much higher among White (56.3/1,000,000) than Black males (10.0/1,000,000), while there was no difference in incidence between White and Black females (3.2/1,000,000). The rates of EGCT among men and women of both races were similar (range:1.9-3.4/1,000,000). The most frequent extragonadal sites were mediastinum among males and placenta among females. The 5-year RSR of testicular GCT was higher among Whites (97%) than Blacks (90%), as was the 5-year RSR of ovarian GCT (Whites, 92%; Blacks 85%). In general, the 5-year RSRs of EGCTs were lower than the 5-year RSRs of GGCTs. The different incidence trends of GGCTs and EGCTs and distinct age-specific incidence patterns by anatomical site of EGCTs suggest that GGCTs and EGCTs may have different aetiologies.

Reactivity of Germ Cell Maturation Stage-specific Markers in Classical and Spermatocytic Seminoma

This paper presents data supporting the model that classical seminoma originates from a primordial germ cell/gonocyte in which maturation is blocked. Although spermatocytic seminoma might also originate from an embryonic germ cell, it retains the capacity to undergo further maturation, including partial meiosis. Moreover, we identified immunohistochemical markers helpful in the diagnosis of spermatocytic seminomas.