Low level expression of human telomerase reverse transcriptase predicts cancer-related death and progression in embryonal carcinoma.

INTRODUCTION: hTERT (human telomerase reverse transcriptase) is a catalytic subunit of the enzyme telomerase and has a role in cell proliferation, cellular senescence, and human aging. MATERIALS AND METHODS: The purpose of this study was to evaluate the expression and significance of hTERT protein expression as a prognostic marker in different histological subtypes of testicular germ cell tumors (TGCTs), including 46 embryonal carcinomas, 46 yolk sac tumors, 38 teratomas, 84 seminomas as well as two main subtypes of seminomas and non-seminomas using tissue microarray (TMA) technique. RESULTS: The results showed that there is a statistically significance difference between the expression of hTERT and various histological subtypes of TGCTs (P < 0.001). In embryonal carcinoma, low level expression of hTERT protein was significantly associated with advanced pT stage (P = 0.023) as well as tunica vaginalis invasion (P = 0.043). Moreover, low level expression of hTERT protein was found to be a significant predictor of worse DSS (log rank: P = 0.011) and PFS (log rank: P = 0.011) in the univariate analysis. Additionally, significant differences were observed (P =0.021, P =0.018) with 5-year survival rates for DSS and PFS of 66% and 70% for moderate as compared to 97% and 97% for high hTERT protein expression, respectively. CONCLUSION: We showed that hTERT protein expression was associated with more aggressive tumor behavior in embryonal carcinoma patients. Also, hTERT may be a novel worse prognostic indicator of DSS or PFS, if the patients are followed up for more time periods.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

OBJECTIVE: DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. METHODS: The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. RESULTS: DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. CONCLUSIONS: Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor.

PARP expression in germ cell tumours.

BACKGROUND: Poly(ADP-ribose)polymerase (PARP) inhibitors represent a new class of promising drugs in anticancer therapy. AIMS: To evaluate PARP expression in testicular germ cell tumours (GCTs) and to correlate expression patterns with clinicopathological variables. METHODS: In this translational study, tumour specimens from 124 patients with GCTs (114 patients with testicular primary tumours and 10 with extragonadal GCTs) were identified. PARP expression was detected by immunohistochemistry using monoclonal antibodies, scored by the multiplicative quickscore (QS) method and compared to PARP expression in normal testicular tissue. RESULTS: We observed higher expression of PARP in testicular tumours compared to normal testicular tissue (mean QS=10.04 vs 3.31, p<0.0000001). Mean QS±SD for each histological subtype was as follows: intratubular germ cell neoplasia unclassified (IGCNU)=18.00±0.00, embryonal carcinoma=9.62±5.64, seminoma=9.74±6.51, yolk sac tumour=7.8±7.20, teratoma=5.87±5.34, and choriocarcinoma=4.50±8.33. The PARP overexpression (QS>9) was most often detected in IGCNU (100% of specimen with PARP overexpression), seminona (52.6%), embryonal carcinoma (47.0%), yolk sac tumour (33.3%), teratoma (26.7%) and choriocarcinoma (25.0%), compared to 1.9% of normal testicular tissue specimens. There was no association between PARP expression and clinical variables. CONCLUSIONS: In this pilot study, we showed for the first time, that PARP is overexpressed in testicular germ cell tumours compared to normal testis.

Skeletal myosin light chain kinase regulates skeletal myogenesis by phosphorylation of MEF2C.

The MEF2 factors regulate transcription during cardiac and skeletal myogenesis. MEF2 factors establish skeletal muscle commitment by amplifying and synergizing with MyoD. While phosphorylation is known to regulate MEF2 function, lineage-specific regulation is unknown. Here, we show that phosphorylation of MEF2C on T(80) by skeletal myosin light chain kinase (skMLCK) enhances skeletal and not cardiac myogenesis. A phosphorylation-deficient MEF2C mutant (MEFT80A) enhanced cardiac, but not skeletal myogenesis in P19 stem cells. Further, MEFT80A was deficient in recruitment of p300 to skeletal but not cardiac muscle promoters. In gain-of-function studies, skMLCK upregulated myogenic regulatory factor (MRF) expression, leading to enhanced skeletal myogenesis in P19 cells and more efficient myogenic conversion. In loss-of-function studies, MLCK was essential for efficient MRF expression and subsequent myogenesis in embryonic stem (ES) and P19 cells as well as for proper activation of quiescent satellite cells. Thus, skMLCK regulates MRF expression by controlling the MEF2C-dependent recruitment of histone acetyltransferases to skeletal muscle promoters. This work identifies the first kinase that regulates MyoD and Myf5 expression in ES or satellite cells.

Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans.

Histone deacetylase (HDAC) inhibitors induce cell cycle arrest, differentiation or apoptosis in tumour cells and are, therefore, promising anti-cancer reagents. However, the specific HDAC isoforms that mediate these effects are not yet identified. To explore the role of HDAC1 in tumourigenesis and tumour proliferation, we established an experimental teratoma model using wild-type and HDAC1-deficient embryonic stem cells. HDAC1-deficient teratomas showed no significant difference in size compared with wild-type teratomas. Surprisingly, loss of HDAC1 was not only linked to increased apoptosis, but also to significantly enhanced proliferation. Epithelial structures showed reduced differentiation as monitored by Oct3/4 expression and changed E-cadherin localization and displayed up-regulated expression of SNAIL1, a regulator of epithelial cell plasticity. Increased levels of the transcriptional regulator SNAIL1 are crucial for enhanced proliferation and reduced differentiation of HDAC1-deficient teratoma. Importantly, the analysis of human teratomas revealed a similar link between loss of HDAC1 and enhanced tumour malignancy. These findings reveal a novel role for HDAC1 in the control of tumour proliferation and identify HDAC1 as potential marker for benign teratomas.

Nucleoside drugs induce cellular differentiation by caspase-dependent degradation of stem cell factors.

BACKGROUND: Stem cell characteristics are an important feature of human cancer cells and play a major role in the therapy resistance of tumours. Strategies to target cancer stem cells are thus of major importance for cancer therapy. Differentiation therapy by nucleoside drugs represents an attractive approach for the elimination of cancer stem cells. However, even if it is generally assumed that the activity of these drugs is mediated by their ability to modulate epigenetic pathways, their precise mode of action remains to be established. We therefore analysed the potential of three nucleoside analogues to induce differentiation of the embryonic cancer stem cell line NTERA 2 D1 and compared their effect to the natural ligand retinoic acid. METHODOLOGY/PRINCIPAL FINDINGS: All nucleoside analogues analyzed, but not retinoic acid, triggered proteolytic degradation of the Polycomb group protein EZH2. Two of them, 3-Deazaneplanocin A (DZNep) and 2'-deoxy-5-azacytidine (decitabine), also induced a decrease in global DNA methylation. Nevertheless, only decitabine and 1beta-arabinofuranosylcytosine (cytarabine) effectively triggered neuronal differentiation of NT2 cells. We show that drug-induced differentiation, in contrast to retinoic acid induction, is caused by caspase activation, which mediates depletion of the stem cell factors NANOG and OCT4. Consistent with this observation, protein degradation and differentiation could be counteracted by co-treatment with caspase inhibitors or by depletion of CASPASE-3 and CASPASE-7 through dsRNA interference. In agreement with this, OCT4 was found to be a direct in-vitro-target of CASPASE-7. CONCLUSIONS/SIGNIFICANCE: We show that drug-induced differentiation is not a consequence of pharmacologic epigenetic modulation, but is induced by the degradation of stem-cell-specific proteins by caspases. Our results thus uncover a novel pathway that induces differentiation of embryonic cancer stem cells and is triggered by the established anticancer drugs cytarabine and decitabine. These findings suggest new approaches for directly targeting the stem cell fraction of human tumours.

Therapeutic potential of Mdm2 inhibition in malignant germ cell tumours.

BACKGROUND: Inadequate response to cisplatin-based chemotherapy is associated with poor prognosis in patients with advanced malignant testicular germ cell tumours (TGCTs), especially of the nonseminomatous type. Novel chemotherapeutic agents have failed so far to significantly improve the outcome of such patients. The majority of these tumours express low levels of p53, and TP53 mutations are rarely observed. Murine double minute 2 (Mdm2) inhibitors enhance apoptosis in tumours harbouring wild-type p53. OBJECTIVE: We sought to investigate the potential therapeutic value of Mdm2 in TGCT-derived cell lines with the histology of nonseminoma. DESIGN, SETTING, AND PARTICIPANTS: The Mdm2 inhibitor nutlin-3 was evaluated alone and in combination with cisplatin in a panel of germ cell tumour (GCT)-derived cell lines (embryonal carcinomas, being the nonseminomatous stem-cell component) with wild-type (NT2 and 2102EP cells) and mutant (NCCIT cells) p53 status. MEASUREMENTS: Biological consequences of Mdm2 inhibition were determined by analysis of the p53 pathway, cell proliferation, and apoptosis. RESULTS AND LIMITATIONS: Nutlin-3 exhibited significant activity (IC50 2.8 µM) in NT2 and 2102EP (wild-type p53) but not in p53-mutant NCCIT cells (<10% inhibition at 10 µM). At concentrations beyond 500 nM, additive effects were seen for the combination of nutlin-3 and cisplatin in NT2 and 2102EP cells but not in NCCIT cells. This correlated with the induction of p53 and its target p21, suggesting an on-target effect of nutlin-3. Moreover, nutlin-3 (5 µM) and cisplatin (0.5 µM) additively induced caspase cleavage and apoptosis in NT2 cells and 2102-EP cells but not in p53-mutant NCCIT cells. CONCLUSIONS: These results provide strong evidence for further development of pharmacologic Mdm2 inhibition for the treatment of patients suffering from high-risk nonseminomatous TGCT with wild-type p53 status.

Neural differentiation potentiated by the leukaemia inhibitory factor through STAT3 signalling in mouse embryonal carcinoma cells.

LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.

Topoisomerase II alpha expression in testicular germ cell tumors.

OBJECTIVES: Inhibitors of topoisomerase II alpha (TopoIIalpha), an enzyme with a crucial role in DNA maintenance, are included in the chemotherapy protocols for testicular germ cell tumors (GCTs). Despite the success of current chemotherapy regimens, a significant number of patients experience relapse. We analyzed TopoIIalpha expression in primary and metastatic testicular GCTs because this enzyme is a target for some antineoplastic agents. METHODS: Primary GCT specimens from 109 patients, including 57 seminomas and 52 mixed GCTs (41 embryonal carcinomas, 23 yolk sac tumors, 19 seminomas, 8 choriocarcinomas, 17 teratomas with immature elements, and 16 teratomas with mature elements), were obtained from our archives. The metastatic lesions from 11 of the patients with mixed GCTs included seven teratomas with mature components, five embryonal carcinomas, one yolk sac tumor, one choriocarcinoma, and one teratoma with immature components. Representative sections were subjected to immunohistochemistry with monoclonal antibody against TopoIIalpha, and the nuclear staining findings were evaluated. RESULTS: Most embryonal carcinoma (100%), yolk sac tumor (95%), seminoma (88%), and choriocarcinoma (62%) components of the GCTs were TopoIIalpha immunoreactive. None of the teratoma specimens with mature elements expressed TopoIIalpha. CONCLUSIONS: The results of our study have shown that TopoIIalpha is expressed in most seminomas, embryonal carcinomas, yolk sac tumors, and choriocarcinomas, suggesting a possible mechanism of sensitivity of these components to TopoIIalpha inhibitors. Teratomas with mature and immature elements expressed low levels of TopoIIalpha, which might contribute to their chemoresistance. These findings imply that the variable chemoresponsiveness of testicular GCTs could have an underlying molecular basis.

Activation-induced cytidine deaminase (AID) is expressed in normal spermatogenesis but only infrequently in testicular germ cell tumours.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes in antigen-dependent B-cell maturation. SHM is not restricted to immunoglobulin gene loci, raising the possibility of a function for AID in other cell types. In this study, it is shown that AID is expressed in spermatocytes in the human testis. AID was mostly cytoplasmic but nuclear AID was also observed in a proportion of cells, in keeping with the DNA deamination model of AID function. Intratubular germ cell neoplasia unclassified (IGCNU), the precursor lesion of testicular cancers, was AID-negative. Seminomas also lacked AID expression. Nuclear and cytoplasmic AID expression was observed in three of 32 mixed non-seminomatous germ cell tumours. The results provide evidence for a physiological role for AID outside the immune system. AID expression in spermatocytes points to a role in meiosis. It remains uncertain whether AID may also contribute to the genetic aberrations characteristically found in testicular germ cell tumours. The consistent absence of detectable AID expression in atypical spermatogonia of IGCNU and its rare expression in germ cell tumours suggest that continued expression of AID is not involved in the pathogenesis of germ cell tumours.

Aneuploidy of human testicular germ cell tumors is associated with amplification of centrosomes.

Testicular germ cell tumors occur in three age groups. Seminomas and nonseminomas of adults, including mature teratomas, and the precursor carcinoma in situ (CIS) are aneuploid. This also holds true for yolk sac tumors of newborn and infants, while the mature teratomas of this age are diploid. In contrast, spermatocytic seminomas occurring in the elderly contain both diploid and polyploid cells. Aneuploidy has been associated with centrosome aberrations, sometimes related to overexpression of STK15. Aneuploidy of non-neoplastic germ cells has been demonstrated in the context of male infertility, a risk factor for the development of seminoma/nonseminoma. We investigated aneuploidy, centrosome aberrations and the role of STK15 in different types of testicular germ cell tumors as well as in normal and disturbed spermatogenesis. The aneuploid seminomas and nonseminomas tumors (including CIS) showed increased numbers of centrosomes, without STK15 amplification or overexpression. Four out of six infantile teratomas had normal centrosomes, the remaining two and an infantile yolk sac tumor showed a heterogeneous pattern of cells with normal or amplified centrosomes. Spermatocytic seminomas had two, four or eight centrosomes. Germ cells in seminiferous tubules with disturbed spermatogenesis shared both aneuploidy and centrosome abnormalities with seminomas/nonseminomas and showed a more intense STK15 staining than those with normal spermatogenesis and CIS. Therefore, aneuploidy of testicular germ cell tumors is associated with amplified centrosomes probably unrelated to STK15.

Involvement of the phosphoinositide 3-kinase/protein kinase B signaling pathway in insulin/IGF-I-induced chondrogenesis of the mouse embryonal carcinoma-derived cell line ATDC5.

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin/insulin-like growth factor-I (IGF-I) stimulation. In the present study, we examined whether insulin/IGF-I stimulation caused activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB) pathway in ATDC5 cells. We also determined whether the insulin-stimulated differentiation of ATDC5 cells into chondrocytes could be mimicked by activation of the PKB pathway alone. ATDC5 cells produced phosphatidylinositol 3,4,5-trisphosphate and the pleckstrin homology domain of PKB was recruited to the plasma membrane in response to insulin stimulation. This was probably a result of activation of PI3K because the PI3K inhibitors, wortmannin and LY294002, inhibited both responses, although the effective concentrations were as high as 10 microM. Insulin stimulation caused the chondrogenic differentiation of ATDC5 cells as assessed by chondrogenic nodule staining with alcian blue. The addition of wortmannin or LY294002, PI3K inhibitors, suppressed the staining, and the suppression was reversible, indicating the effect of the inhibitors is not toxic. Finally, we exogenously expressed a constitutively-activated from of PKB (myristoylated PKB, myr-PKB) in ATDC5 cells, and found the chondrogenic differentiation of ATDC5 cells to form nodules occurred in the absence of insulin stimulation. The kinase-negative mutant of myr-PKB did not caused differentiation, indicating that kinase activity is required. These results support the hypothesis that the PI3K/PKB signaling pathway is involved in the chondrogenic differentiation of ATDC5 cells in response to insulin/IGF-I stimulation. This is the first report that demonstrates the involvement of phosphoinositide signaling in the induction of chondrogenesis from undifferentiated cells.

Serum lactate dehydrogenase isoenzyme 1 and relapse in patients with nonseminomatous testicular germ cell tumors clinical stage I.

Serum lactate dehydrogenase isoenzyme 1 catalytic concentration (S-LD-1) was measured at the time of orchiectomy in 104 patients with nonseminomatous testicular germ cell tumors (NSTGCT) clinical stage I who participated in a randomized study comparing surveillance after orchiectomy (group I) and radiotherapy (group II). For 68 patients, S-LD-1 was measured in a serum sample before or on the day of the orchiectomy. Twenty-seven patients (40%) had elevated S-LD-1; median 102 U/L (range 41-335). For the remaining 36 patients. S-LD-1 was measured in a serum sample after orchiectomy: 8 of these patients (22%) had elevated S-LD-1. S-LD-1 was normalized shortly after surgery in most patients with a preorchiectomy elevated S-LD-1. Fifteen of the 68 patients relapsed: 9 out of 27 with an elevated S-LD-1 and 6 out of 41 patients with normal S-LD-1 (p = 0.13, Fisher's exact test). In group 1, those with a preoperatively elevated S-LD-1 had a lower 8-years' relapse-free survival than those with a normal S-LD-1 (40% vs. 80%, p = 0.003, log-rank test). The role of S-LD-1 in the staging, prognostication and monitoring of patients with NSGCT clinical stage I should be further explored in a large, prospective study.

Activation of c-Jun amino-terminal kinase is required for retinoic acid-induced neural differentiation of P19 embryonal carcinoma cells.

P19 embryonal carcinoma cells are known to differentiate into neurons and glia when treated with relatively high concentrations (>100 nM) of retinoic acid (RA). Concomitant with this RA-induced neural differentiation, we observed an activation of the c-Jun amino-terminal kinase (JNK). JNK was required for the RA-induced neural differentiation, because dominant-negative JNK blocked the differentiation. Studies using protein phosphatase inhibitors and protein kinase inhibitors suggested that both okadaic acid-sensitive protein phosphatase(s) and protein kinase C participate in the RA-induced activation of JNK.

Disassociation of MAPK activation and c-Fos expression in F9 embryonic carcinoma cells following retinoic acid-induced endoderm differentiation.

Retinoic acid induces cell differentiation and suppresses cell growth in a wide spectrum of cell lines, and down-regulation of activator protein-1 activity by retinoic acid contributes to these effects. In embryonic stem cell-like F9 teratocarcinoma cells, which are widely used to study retinoic acid actions on gene regulation and early embryonic differentiation, retinoic acid treatment for 4 days resulted in suppression of cell growth and differentiation into primitive and then visceral endoderm-like cells, accompanied by a suppression of serum-induced c-Fos expression. The MAPK (ERK) pathway was involved in mitogenic signaling in F9 cells stimulated with serum. Surprisingly, although c-Fos expression was reduced, the MAPK activity was not decreased by retinoic acid treatment. We found that retinoic acid treatment inhibited the phosphorylation of Elk-1, a target of activated MAPK required for c-Fos transcription. In F9 cells, the MAPK/MEK inhibitor PD98059 suppressed Elk-1 phosphorylation and c-Fos expression, indicating that MAPK activity is required for Elk-1 phosphorylation/activation. Phosphoprotein phosphatase 2B (calcineurin), the major phosphatase for activated Elk-1, is not the target in the disassociation of MAPK activation and c-Fos expression since its inhibition by cyclosporin A or activation by ionomycin had no significant effects on serum-stimulated c-Fos expression and Elk-1 phosphorylation. Thus, we conclude that retinoic acid treatment to induce F9 cell differentiation uncouples Ras/MAPK activation from c-Fos expression by reduction of Elk-1 phosphorylation through a mechanism not involving the activation of phosphoprotein phosphatase 2B.

Syk protein-tyrosine kinase is involved in neuron-like differentiation of embryonal carcinoma P19 cells.

Syk has been implicated in activated immunoreceptors to downstream signaling events in hematopoietic cells. Here we report that Syk is expressed in neuron-like cells and involved in neuron-like differentiation of embryonal carcinoma P19 cells. Immunoblot, RT-PCR, and Northern analysis indicated that Syk is expressed in mouse brain, PC12 and P19 cells. In addition, Syk was found to be tyrosine phosphorylated during neuron-like differentiation of P19 cells. Furthermore, adenovirus-mediated overexpression of Syk induced supernumerary neurite formation and extracellular signal-regulated kinase (ERK) activation in P19 cells. These results suggest that Syk plays an important role in signaling steps leading to ERK activation in P19 cells.

Activation of TrkA tyrosine kinase in embryonal carcinoma cells promotes cell compaction, independently of tyrosine phosphorylation of catenins.

Cadherins are transmembrane receptors whose extracellular domain mediates homophilic cell-cell interactions, while their cytoplasmic domain associates with a family of proteins known as catenins. Although the mechanisms that regulate the assembly and functional state of cadherin-catenin complexes are poorly understood, current evidence supports a role for protein tyrosine kinase activity in regulating cell adhesion and migration. Tyrosine phosphorylation of catenins is thought to mediate loss of intercellular adhesion promoted by activation of receptor tyrosine kinases in epithelial cells. Here, we show that activation of ectopically expressed TrkA, the tyrosine kinase receptor for nerve growth factor (NGF), stimulates embryonal carcinoma P19 cells to develop extensive intercellular contacts and to assemble into closely packed clusters. Thus, activation of receptor tyrosine kinases can differentially regulate adhesiveness by cell-type-specific mechanisms. Furthermore, activation of TrkA in P19 and epithelial MDCK cells induces tyrosine phosphorylation of p120(ctn) and of beta-catenin, irrespective of the elicited cellular response. The selective Src tyrosine kinase inhibitor PP2, however, suppresses NGF- or HGF-induced tyrosine phosphorylation of catenins in both P19 and MDCK cells without interfering with the acquisition of a compacted or scattered phenotype. These findings provide a cogent argument for considering that tyrosine phosphorylation of catenins is dispensable for their interaction with cadherins and, ultimately, for the modulation of cadherin-based cell adhesion by receptor tyrosine kinases.

Somatic isoform of angiotensin I-converting enzyme in the pathology of testicular germ cell tumors.

Retained fetal expression of angiotensin I-converting enzyme (ACE, CD143) has recently been shown in intratubular germ cell neoplasms (IGCN) and invasive germ cell tumors (GCT), suggesting the somatic isoform (sACE) as a characteristic component of neoplastic germ cells. We analyzed the distribution of sACE in 159 testicular GCT, including 87 IGCN. sACE protein was determined by immunohistochemistry (MAb CG2) on routinely formalin-fixed and paraffin-embedded tissue sections, supplemented by mRNA expression analysis using in situ hybridization. These data were compared with those obtained by germ cell/placental alkaline phosphatases (PIAP; MAbs PL8-F6 and 8A9) employing an uniform score system for the evaluation of immunoreactivity (IRS; possible values from 0 to 12). Expression of sACE and PIAP was found in all 87 analyzed IGCN (IRS > 4, median IRS of 12). Heterogeneous staining patterns were not related to the type of adjacent GCT but correlated with low expression in adjacent seminomas (P =.032 for sACE; P =.005 for PIAP). Both sACE and PIAP often showed a decreased and more heterogeneous but still moderate expression in 91 classic seminomas (median IRS of 8) and were completely absent in tumor cells of spermatocytic seminomas. Despite all similarities, we found sACE and PIAP differently regulated during GCT progression. This was documented by a well-preserved expression of either sACE or PIAP or both in all classic seminomas, low PIAP immunoreactivity in metastasis of seminomas, and completely diverging expression patterns in nonseminomatous GCT. Our findings underline the close molecular relationship between IGCN and seminoma, and suggest sACE as an appropriate marker for seminomatous differentiated tumors. HUM PATHOL 31:1466-1476.

Expression of the RNA component of human telomerase in adult testicular germ cell neoplasia.

BACKGROUND: During human development, telomerase is repressed in most somatic cells, whereas it is maintained in male germline cells. Reactivation of telomerase has been associated with somatic cancers. To the authors' knowledge, the role of telomerase in germ cell derived malignancies has not previously been evaluated. METHODS: A radioactive in situ hybridization method was used to study the expression of the RNA component of human telomerase (hTR) in 22 cases of adult testicular germ cell neoplasia encompassing all major histomorphologic types. For precise cell identification, hTR in situ hybridization was combined with immunohistochemistry in select cases. RESULTS: Testicular germ cell tumors showed differential expression of hTR. The highest level of expression was seen in embryonal carcinoma. Seminoma and unclassified intratubular germ cell neoplasia exhibited moderate levels of expression. Yolk sac tumor was characterized by a range of expression, which mirrored its morphologic variation. Immature teratoma recapitulated the down-regulation of telomerase manifested during human embryogenesis. Mature teratoma represented the adult pattern of somatic repression. Notably, choriocarcinoma showed modest expression. The expression of spermatocytic seminoma was intermediate between that of classic seminoma and embryonal carcinoma. No difference in expression was evident between matching intratubular and invasive components. In nonneoplastic testis, hTR expression was down-regulated during spermatogenesis and was absent in spermatozoa. Expression was negligible in rete testis and interstitial Leydig cells, and low in epididymis. Unexpectedly, Sertoli cells, which are testicular accessory somatic cells, displayed the most intense expression observed in this study. CONCLUSIONS: In testicular germ cell tumors of young adults (and during spermatogenesis), hTR expression is down-regulated with differentiation, irrespective of the aggressiveness of the tumors. Spermatocytic seminoma, regarded as a low grade malignancy, shows moderately intense expression.