N-cadherin expression in malignant germ cell tumours of the testis

Background Testicular germ cell tumours (TGCTs) are the most common malignancy in young men aged 18–35 years. They are clinically and histologically subdivided into seminomas and non-seminomas. Cadherins are calcium-dependent transmembrane proteins of the group of adhesion proteins. They play a role in the stabilization of cell-cell contacts, the embryonic morphogenesis, in the maintenance of cell polarity and signal transduction. N-cadherin (CDH2), the neuronal cadherin, stimulates cell-cell contacts during migration and invasion of cells and is able to suppress tumour cell growth. Methods Tumour tissues were acquired from 113 male patients and investigated by immunohistochemistry, as were the three TGCT cell lines NCCIT, NTERA-2 and Tcam2. A monoclonal antibody against N-cadherin was used. Results Tumour-free testis and intratubular germ cell neoplasias (unclassified) (IGCNU) strongly expressed N-cadherin within the cytoplasm. In all seminomas investigated, N-cadherin expression displayed a membrane-bound location. In addition, the teratomas and yolk sac tumours investigated also differentially expressed N-cadherin. In contrast, no N-cadherin could be detected in any of the embryonal carcinomas and chorionic carcinomas examined. This expression pattern was also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma. Conclusions N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT.

Cadherins are Ca 2+ -dependent transmembrane glycoproteins belonging to the group of adhesion molecules.
More than 80 different members constitute the group of cadherins, such as the well-investigated epithelial, neural and placental cadherins [4]. Cadherins are considered to play a major role in cell-cell contacts, in the development of different organs and also in the genesis of tumours. Furthermore, they function as metastasissuppressing proteins [5]. Decreased cadherin expression is normally found in cancers and is associated with an increased rate of metastasis [6].
N-cadherin (CDH2), the neuronal cadherin, is a 140 kD protein and was first identified in mouse brain tissue [7]. It plays an important role in migration, differentiation, embryonic development and metastatic behaviour of tumour cells [8].The function of N-cadherin is dependent on its association with the actin-cytoskeleton, which is mediated through interactions between the C-terminal region of N-cadherin and the cytoplasmic catenin proteins [9,10]. N-cadherin has been reported to be expressed in different normal tissues [11]. Furthermore, N-cadherin expression could be identified in benign and malignant neoplastic tissues of epithelial and mesenchymal origin [12][13][14][15][16][17]. In the present study we analysed the expression of N-cadherin in testicular germ cell tumours.

Tissue samples of primary TGCT
Tumour tissues from orchiectomy specimens were acquired from 113 male patients from the University Medical Centre Göttingen, Germany (mean age = 33.86 years). Tumours were classified and staged on the basis of the WHO classification [18]. In the present study a number of 123 blocks have been tested. Investigated cases included IGCNU (n=20), seminomas (n= 77), embryonal carcinomas (n= 40), teratomas (n=17), chorionic carcinomas (n=4), and yolk sac tumours (n=11). One section was made of each tumour per 0.5 cm tumour diameter. Tumour tissues from each testis were immediately fixed in formalin and embedded in paraffin. In addition, normal testis specimens were analysed (n=28, mean age 35.82 ± 12.41). Ethical approval for using the human material in the present study was obtained from the Ethics Committee of the University Medical Centre Göttingen.
Two independent investigators evaluated all tissue sections considering membranous and cytoplasmic N-cadherin staining and using an immunoreactive staining score (IRS). The percentage of positively stained cells was first categorized using a 0-4 scoring system: Score 0 = 0% positive cells, score 1= less than 10% positive cells, score 2 = 10-50% positive cells, score 3 = 51-80% positive cells and score 4 = > 80% positive cells. The intensity of staining was evaluated on a graded scale (0 = negative; 1 = weak; 2 = intermediate; 3 = strong). For the final IRS, the scores of intensity and staining were multiplied and the mean value per patient was calculated.

Immunohistochemistry
Immunohistochemical reactions were performed on 5-μm formalin-fixed and paraffin-embedded testis tissue sections of the testis. The sections were incubated in citrate buffer (pH 6). For primary antibodies the sections were incubated for 30 minutes at room temperature with monoclonal antibody against N-cadherin diluted at 1:50 (DAKO, Hamburg, Germany). Thereafter the sections were incubated with the biotinylated secondary antibody and the streptavidin alkaline phosphatase (REAL, Dako, Hamburg, Germany). Fast red (Dako) was applied to visualise the sites of immunoprecipitations. Tissue samples were analysed by light microscopy after counterstaining with Meyer's haematoxylin.

Results
Immunohistochemical staining for N-cadherin in tumour-free testis, IGCNU and invasive TGCT Tumour-free testes exhibit strong cytoplasmatic expression of N-cadherin in all spermatogenesis and Sertoli cells    All examined seminomas show a membrane-bound N-cadherin expression, with the expression located cytoplasmatically in only some cases (IRS 9.38 ± 2.59) (Figure 2A + B).
All investigated teratomas show diverse N-cadherin expression pattern. Areas with primitive neuronal elements display positive N-cadherin staining (IRS 7.11 ± 0.99).
Other tumour components like hyaline cartilage or connective tissue are negative for N-cadherin expression ( Figure 3A-D). Yolk sac tumours show a strong N-cadherin expression pattern within the tumour cells. The expression is located cytoplasmatically or bound to the membrane (IRS 9.90 ± 0.94) ( Figure 3E).
In contrast, N-cadherin can not be detected in any of the embryonal carcinomas examined; this is also seen in the investigated mixed tumours consisting of seminomas, teratomas, and embryonal carcinoma ( Figure 4A-D). Chorionic carcinoma components within malignant germ cell tumours prove also to be negative for N-cadherin expression ( Figure 4E-F). The immunohistochemical results are summarized in Table 1.

N-cadherin expression in TGCT cell lines
The tumour cell line NCCIT originating from a teratocarcinoma shows strong cytoplasmic N-cadherin expression based on the positive expression in teratomas in vivo. In addition, the Tcam2 cell line originating from a seminoma, also show N-cadherin expression comparable to the in vivo results. NTERA-2 orginating from an embryonal carcinoma, on the other hand, does not show N-cadherin expression as it does in the embryonal carcinoma in vivo ( Figure 5A-C).

Discussion
In young men aged 18 to 35 years, testicular germ cell tumours are the most common malignant tumours. TGCT can be divided into the two groups of seminomas and non-seminomas [1]. Differentiating between seminomas and non-seminomas and also mixed germ cell tumours is important because of their different prognosis and therapy [2].
N-cadherin plays a crucial role in organ development and is usually expressed in neuronal tissue [19]. It could be shown that a non-tissue-specific expression of Ncadherin in tumours plays a crucial role for cell migration, invasion and metastases [20]. This process is also essential in embryonic development and is called epithelial-mesenchymal transition (EMT) [21]. The EMT stimulates the mobility and invasive capacity of tumour cells and it is associated with tumour progression [22,23] as well as the development of chemotherapy resistance [24,25]. In addition, not only a non-tissue-specific expression of N-cadherin, but also the switch from different cadherins to N-cadherin are connected with a poor prognosis [26,27].
In the present study we observed cytoplasmatic and membranous N-cadherin expression in tumour-free testis, IGCNU, seminomas, yolk sac tumours and in primitive neuronal elements within teratomas. Loojenga et.al and   [28,29]. The positive N-cadherin expression in our investigation could be an indication of EMT at least in seminomas. Interestingly, N-cadherin was not expressed in embryonic carcinomas and in components of chorionic carcinoma, as we could show in the present study. In contrast, it could be shown that E-cadherin is expressed in embryonic carcinomas [28]. Hart et al. described a model of tumorgenesis in testis germ cell tumours [30]. In this model non-seminomatous germ cell tumours can arise from IGCNU or from seminomas. It seems that malignant germ cell tumours of the testis without N-cadherin but with positive E-cadherin expression show a differentiation into an embryonic carcinoma or chorionic carcinoma. It would be interesting to see whether the expression of N-cadherin is accompanied by the activation of a corresponding pathway or influence a specific differentiation. Such an analysis would possibly offer insights into the relation between N-cadherin expression and behaviour of the tumour. Several immunohistochemical markers are known for the differential diagnosis of malignant germ cell tumours. Seminomas are usually positive for placental alkaline phosphatise (PLAP) and CD117 (c-kit) [31][32][33]. OCT 4 is expressed in seminomas and embryonal carcinomas [34]. Immunohistochemical expression of alpha fetoprotein (AFP) can be used for the diagnosis of yolk sac tumours [35]. Chorionic carcinoma shows an expression of human chorionic gonadotropin [36]. For embryonal carcinoma CD30 positivity in connection with the expression of CD117 usually verifies the diagnosis. Thus N-cadherin expression correlates with the expression pattern of immunohistochemical markers of malignant germ cell tumours such as CD117, PLAP and AFP. Interestingly CD30 and ßHCG are conversely expressed to N-cadherin.

Conclusions
N-cadherin expression can be used to differentiate embryonal carcinomas and chorionic carcinomas from other histological subtypes of TGCT. Thus, loss of N-cadherin possibly influences the differentiation of malignant germ cell tumours.