OCT4: A sensitive and specific biomarker for intratubular germ cell neoplasia of the testis.

PURPOSE: OCT4 (POU5F1, OCT3) immunostaining highlights pluripotent cells (embryonal carcinoma and seminoma) in primary testicular germ cell tumors, but its relative usefulness in diagnosing intratubular germ cell neoplasia, unclassified (IGCNU) is not well established. The present study aimed to establish OCT4 as a sensitive and specific maker for IGCNU, a putative precursor for adult germ cell tumors. EXPERIMENTAL DESIGN: We evaluated OCT4 immunostaining in 44 cases of IGCNU from patients who had testicular germ cell tumors. In addition, 27 of the 44 IGCNU sections were also examined with antibodies to placenta-like alkaline phosphatase, the most frequently used immunohistochemical marker for intratubular germ cell neoplasia. Sections from the testes of 10 patients who had undergone orchiectomy for hormonal treatment of prostate cancer and from autopsies of 10 patients without histories of germ cell tumors were also examined for OCT4 immunostaining. The immunoreactivity of the autopsy tissues was determined with vimentin staining, and all were reactive. RESULTS: In all 44 of the cases, antibody to OCT4 marked the nuclei of nearly all of the dysplastic cells of intratubular germ cell neoplasia but not non-neoplastic testicular cells. The staining intensity was strong in every case, and there was little or no background staining. All 20 of the control specimens (10 orchiectomy specimens from prostate cancer patients and 10 testes from autopsies) were completely negative for OCT4. The 27 cases that were stained with antiplacenta-like alkaline phosphatase antibodies showed staining of variable intensity in the areas of intratubular germ cell neoplasia, and there was a high level of background staining artifact. CONCLUSIONS: OCT4 is a sensitive and specific maker for intratubular germ cell neoplasia.     

Alkaline phosphatase isozymes in human testicular germ cell tumors, their precancerous stage, and three related cell lines

The four known isozymes of the human alkaline phosphatase (ALP) were detected by isoelectric focusing in extracts of various types of germ cell tumors, three related cell lines, and their precancerous elements (atypical germ cells). In seminoma, placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (PLAP-like) could be separated by isoelectric focusing following isolation by immunoaffinity. The occurrence of both isozymes in seminoma could explain partial heat sensitivity and variation in electrophoretic patterns of the seminoma isozyme frequently observed upon starch gels, in comparison to the normal placental phenotype. The four ALP isozymes are produced not only in germ cell tumors, but already in precancerous tissues. Quantitative analysis showed that the amount of the four isozymes varies in parallel in the tumors tested. Maximal expression was found in seminoma. The relation between ALP gene overexpression and gene amplification by polyploidy of chromosomes 1 and 2 in these lesions is discussed. On the other hand, the ectopic expression of intestinal alkaline phosphatase and PLAP associated with overexpression of PLAP-like in tumor cells as well as in their precancerous stage indicates gene activation by some unknown mechanisms, probably a regulatory process affecting the three tissue-specific ALP genes simultaneously.     

Immunohistochemistry of germ cell and trophoblastic neoplasms

The immunoprofiles of 121 germ cell and trophoblastic neoplasms were defined, using a battery of antibodies against cytokeratin (CK), vimentin (VIM), epithelial membrane antigen (EMA), placental alkaline phosphatase (PLAP), S-100 protein, leukocyte common antigen (LCA), UCHL-1, LN-2, carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), chromogranin A, Leu-7, alpha-fetoprotein (AFP), alpha-1-antitrypsin (AAT), and the beta subunit of human chorionic gonadotropin (BHCG). In addition to 85 neoplasms of testicular origin, the cases included eight ovarian germ cell tumors and 28 extragonadal neoplasms. All tissues had been subjected to formalin fixation and paraffin embedding. Similar immunoreactivity patterns were seen in gonadal and extragonadal neoplasms, gestational and nongestational choriocarcinomas, components of mixed germ cell tumors and their pure counterparts, and metastatic and primary lesions. Placental alkaline phosphatase was a sensitive marker of germ cell differentiation, and expression of this marker in the absence of EMA appeared to be a staining pattern unique to germ cell tumors. Both LCA and S100 were absent in neoplastic germ cells, and thus were useful in differentiating these tumors from malignant lymphoma and malignant melanoma, respectively. Cytokeratin was helpful in distinguishing seminomas/dysgerminomas from nonseminomatous germ cell tumors, although 10% of seminomas showed focal or diffuse cytokeratin reactivity. Finally, 75% of all germ cell neoplasms displayed NSE, calling the specificity of this determinant into question.     

A Putative Mechanism for the Non-Specific Uptake of Intact Radiolabelled Monoclonal Antibodies in the Testes and Prostate

Non-specific testicular accumulation of radiolabeled intact anti-CEA monoclonal antibody (MAb), (A431/26, Behringwerke AG) was observed in 11 out of 12 patients with the testes and prostate included in the examination field at radioimmunoscintigraphy (RIS). Previous studies have shown that placental alkaline phosphatase (PLAP) serves as an Fc-receptor, mediating IgG transport through the placenta. A closely related protein, the germ cell alkaline phosphatase (GCAP), is expressed in the testes. The testicular uptake of IgG is observed only when intact but not fragmented MAbs are used, indicating involvement of Fc-receptors. MDCK cells (dog kidney cell line) transfected with the plasmid pSVT7 containing the GCAP gene were shown to acquire the capacity to both express membrane bound GCAP and to bind IgG on the cell surface. This might indicate that GCAP is responsible for the non-specific accumulation of intact MAb in the testes and prostate often observed when intact murine MAbs are used for radioimmunolocalization (RIL)     

Expression of a specific mouse germ cell nuclear antigen (GCNA1) by early embryonic testicular teratoma cells in 129/Sv-Sl/+ mice

Spontaneous testicular teratomas which develop at a high rate in 129/Sv-Sl/+ mice are thought to be derived from germ cells. The teratomas present initially as groups of atypical germ-like cells within seminiferous cords of the 15.5 days post coitum (dpc) embryonic testes. These pluripotent teratoma stem cells are capable of differentiating into many kinds of tissues in adult mice. In this immunohistochemical study, we have examined the testes of 129/Sv-Sl/+ mice to determine whether the teratoma cells which developed in these gonads retain the nuclear antigen GCNA1. GCNA1 is a 110 kDa mouse Germ Cell Nuclear Antigen recognized by a rat monoclonal antibody 10D9G11. GCNA1 is expressed in mouse germ cells after they migrate into the genital ridge (11.5 dpc), throughout embryonic development until postnatally germ cells arrive at the diplotene/dictyate stage of the first meiotic division, when it is no longer expressed. Early foci (16.5 dpc) of teratoma stem cells in 129/Sv-Sl/+ mice strongly express GCNA1, but down regulate GCNA1 expression by 19.5 dpc. The loss of GCNA1 expression from teratoma stem cells late in embryonic development is in contrast to embryonic gonocytes which retain GCNA1 expression throughout fetal development. All postnatal undifferentiated and differentiated teratoma cells did not appear to express GCNA1. The expression of the germ cell specific nuclear antigen GCNA1 in early teratoma stem cells further demonstrated that the testicular teratomas originate from early germ cells. The stronger reaction of monoclonal antibody 10D9G11 to GCNA1 within early teratoma cells compared to normal germ cells makes GCNA1 useful in identifying early embryonic tumor foci.     

Extragonadal retroperitoneal germ cell tumor: evidence of origin in the testis.

BACKGROUND: The origin of extragonadal retroperitoneal germ cell tumors remains controversial. Whether they develop primarily in the retroperitoneum or whether they are metastases of a primary testicular tumor has long been debated. PATIENTS AND METHODS: We retrospectively analyzed 26 patients treated as having primary extragonadal retroperitoneal germ cell tumors based upon the findings of testicular palpation by the referring physician. Testicular evaluation was then extended with ultrasonographical and histological examinations. RESULTS: Biopsy of the extragonadal tumor was performed in 25 patients, confirming diagnosis of extragonadal retroperitoneal germ cell tumor. Prior to treatment patients were clinically evaluated by several physicians and the testes were not considered suspicious for testicular cancer. At urological workup, testes were found to be atrophic and/or indurated in 14 (54%) patients, enlarged in one (4%) and unremarkable in 11 (42%). Ultrasound examination of the testes in 20 patients showed pathological findings in all of them. Histology of the testis was available in 25 of 26 patients and revealed active tumor in three, intratubular germ cell neoplasia in four, scar tissue in 12, sclerosis in three, sclerosis and fibrosis in one, and fibrosis alone in two. CONCLUSIONS: So-called primary extragonadal germ cell tumors in the retroperitoneum are very likely a rare or non-existing entity and should be considered as metastases of a viable or burned-out testicular cancer until proven otherwise. All of our patients with histologically examined testes had pathological finding, 76% of which were either viable tumor or scars.     

Presence of the rare D-variant heat-stable, placental-type alkaline phosphatase in normal human testis

In 11 adult testes studied, about 0.3 to 4.6% of the total alkaline phosphatase activity was heat stable and L-phenylalanine sensitive but L-homoarginine insensitive. The testicular heat-stable enzyme was more susceptible to inhibition by L-leucine and ethylenediaminetetraacetate than were the normal placental and intestinal enzymes. By antibody-directed enzyme inhibition test, the testicular heat-stable enzyme cross-reacted completely with normal placental enzyme but clearly distinguished itself from a heat-stable component of normal intestinal enzyme. Thus, placental alkaline phosphatase D-variant is synthesized in testis, indicating that the gene for elaborating this placental protein is probably already active in the testicular cells. The high incidence of this protein in cancers of testis and ovary is probably due to its increased production by gonadal genes present in the genome of these particular tumors.     

Intratubular germ cell neoplasia in testicular teratomas and epidermoid cysts. Correlation with prognosis and possible biologic significance

The clinicopathologic features of 4 testicular teratomas in infants, 13 testicular epidermoid cysts in adults, and 8 pure teratomas in adults were compared. Intratubular germ cell neoplasia (ITGCN, carcinoma in situ) was observed in 88% of the teratomas in adults; 63% of the patients in this group had metastases and 37% died. No ITGCN was observed in infant testes with teratomas or adult testes with epidermoid cysts; all of the neoplasms in the latter two groups behaved as benign tumors. ITGCN is associated with malignant potential (P = 0.0004; Fisher's exact test). These tumors comprise a spectrum of clinicopathologic entities that probably reflect differences in pathogenesis.     

MAGE-A4, a germ cell specific marker, is expressed differentially in testicular tumors.

BACKGROUND: Testicular germ cell tumors are the most common malignancy in young males, and the frequency of these tumors has risen dramatically over the last century. Because it is known that the MAGE genes are expressed in a wide variety of tumors but are expressed only in the mitotic spermatogonia (germ cells) and in the primary spermatocytes in the normal testis, the authors screened the expression of MAGE-A4 in a panel of testicular germ cell tumors. METHODS: Monoclonal antibody 57B raised against MAGE-A4 was tested immunohistochemically on 12 classical seminomas, 5 anaplastic seminomas, 10 various specimens of nonseminomatous germ cell tumors (NSGCTs), 2 combined tumors containing seminoma components, 1 Sertoli cell tumor, 2 Leydig cell tumors, and 15 carcinomas in situ (CIS). In addition, monoclonal antibody 57B was tested on embryonic gonad (age 8 weeks) and fetal gonads (ages 15 weeks, 17 weeks, and 28 weeks). RESULTS: Classical seminomas uniformly and specifically expressed MAGE-A4 compared with anaplastic seminomas and NSGCTs, which were negative for this antigen. Specific expression of MAGE-A4 also was seen in subpopulations of CIS cells, providing additional evidence for heterogeneity of the phenotype of these cells, in which it is believed that differentiation and proliferation generate seminomas and NSGCTs. Finally, MAGE-A4 was expressed in the fetal precursors of the stem germ cells from 17 weeks of gestation onward, in accordance the fact that CIS can arise from prespermatogonia in the fetus. CONCLUSIONS: MAGE-A4 can be considered a potential specific marker for normal premeiotic germ cells and germ cell tumors and can be used to characterize classical seminomas.     

Isolation and characterization of differentially expressed genes in invasive and non-invasive immortalized murine male germ cells in vitro

In an attempt to elucidate the potential of premeiotic male germ cells to malignant transformation both the invasiveness and the differential gene expression of several putative tumor markers of the spermatogonia-derived cell line GC-1spg and the spermatocyte-derived cell line GC-4spc were analyzed. Studies, using RT-PCR analysis, of the expression pattern of the alkaline phosphatase isoenzymes which serve as markers for testicular germ cell tumors demonstrated that the expression of the endogenous mouse embryonic alkaline phosphatase (EAP) is upregulated in the GC-1spg cell line. Additionally, after transfection of GC-1spg cells and GC-4spc cells with a GCAP-CAT construct, an increased promoter activity of the human germ cell alkaline phosphatase (GCAP), the equivalent human isoenzyme of EAP, was shown in GC-1spg. Furthermore, an in vitro Matrigel invasion assay revealed a significant higher invasive potential of GC-1spg cells as compared to GC-4spc cells. Finally, a suppression subtractive hybridization on RNA of invasive GC-1spg cells and non-invasive GC-4spc cells was performed. In total, 31 cDNA sequences were isolated and further analyzed. Among these, 18 known sequences and 13 unknown sequences were determined. Northern blot analysis revealed that one unknown gene and eight known genes, namely integrin alpha 6, L6 antigen, annexin VIII, BVL-1 retrotransposon, protective protein, replacement variant histone 3.3, alpha-catenin and LPS-binding protein, are over-expressed in invasive GC-1spg cells. Taken together, both the enhanced invasive activity of GC-1spg cells and the upregulated expression of genes involved in the process of tumor progression suggest that the immortalized spermatogonia-derived cell line GC-1spg does have a higher potential to malignant transformation than the immortalized spermatocyte-derived cell line GC-4spc.     

Synchronous bilateral primary germ cell tumors of the testis: a case report and review of the literature

Synchronous bilateral primary germ cell tumors of the testis are exceedingly rare. The most common synchronous testicular tumors are seminomas, followed by embryonal carcinomas, teratocarcinomas, and choriocarcinomas. In a series of 385 patients we found nine with bilateral primary germ cell tumors of the testis (2.3%), including one with synchronous involvement of both testes. The treatment of synchronous bilateral primary germ cell tumors of the testis is in principle the same as that of solitary testicular primary germ cell tumors, and is based on tumor histology and tumor metastasis.     

Burned-out testicular tumor: a case report

Germ cell tumors constitute the majority of all testicular tumors, which are relatively rare overall and are mainly encountered in young adults and teenagers. The term 'burnedout' germ cell tumor refers to the presence of a metastatic germ cell tumor with histological regression of the primary testicular lesion. Clinical examination of the testes and scrotal sonography is pivotal in the initial diagnosis of such neoplasms. We present a case of a 31-year-old male with a retroperitoneal mass and no palpable lesion on testicular examination.     

Molecular characterization of hiwi,a human member of the piwi gene family whose overexpression is correlated to seminomas

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Drosophila melanogaster and C. elegans to Arabidopsis. Here we report the molecular characterization of hiwi, a human member of the piwi gene family. hiwi maps to the long arm of chromosome 12, band 12q24.33, a genomic region that displays genetic linkage to the development of testicular germ cell tumors of adolescents and adults (TGCTs), i.e., seminomas and nonseminomas. In addition, gain of this chromosomal region has been found in some TGCTs. hiwi encodes a 3.6 kb mRNA that is expressed abundantly in the adult testis. It encodes a highly basic 861-amino-acid protein that shares significant homology throughout its entire length with other members of the PIWI family proteins in Drosophila, C. elegans and mammals. In normal human testes, hiwi is specifically expressed in germline cells, with its expression detectable in spermatocytes and round spermatids during spermatogenesis. No expresssion was observed in testicular tumors of somatic origin, such as Sertoli cell and Leydig cell tumors. Enhanced expression was found in 12 out of 19 sampled testicular seminomas-tumors originating from embryonic germ cells with retention of germ cell phenotype. In contrast, no enhanced expression was detected in 10 nonseminomas-testicular tumors that originate from the same precursor cells as seminomas yet have lost their germ cell characteristics. Finally, no enhanced expression was detected in four spermatocytic seminomas-testicular tumors that most likely originate from germ cells capable of partial meiosis. Thus, hiwi is specifically expressed in both normal and malignant spermatogenic cells in a maturation stage-dependent pattern, in which it might function in germ cell proliferation.     

Cryopreservation and transplantation of spermatogonia and testicular tissue for preservation of male fertility

The existence of spermatogonial stem cells in the testis offers clinically relevant options for preservation and restoration of male fertility. New approaches based on male germ cell transplantation and testicular tissue grafting can be applied to generate a limited number of sperm cells and could therefore be considered important new avenues for restoration of fertility in oncological patients. We have developed approaches to infuse germ cells into rodent and primate testes and shown that germ cell transplantation is a procedure for restoration of spermatogenesis in the testis that might be adaptable to primates. As a promising alternative, grafting of testicular tissue has been used to produce fertile sperm. The rapid progress in the development of novel experimental strategies to generate sperm by transplantation of spermatogonial stem cells or by grafting of testicular tissue should stimulate oncologists to consider the cryopreservation of testicular tissue. This review introduces the reader to the physiology of spermatogonial stem cells and summarizes the current and potential future options for fertility preservation in male oncological patients.     

Intratesticular transplantation of testicular cells from leukemic rats causes transmission of leukemia

A rat T-cell leukemia model was used to study the safety of germ cell transplantation as a mean of preventing infertility in males undergoing gonadotoxic cancer treatment. Donor germ cells were harvested from the testes of terminally ill leukemic rats and were either used directly or cryopreserved and thawed before transplantation by rete testis microinjection. All rats transplanted with testicular cells from leukemic donors developed signs of terminal rat T-cell leukemia, whereas control animals remained healthy. Cryopreservation of the donor germ cells caused a 3- to 6-day delay in the terminal phase of leukemia. When a known number of leukemic cells were mixed with germ cells and microinjected into the testis, the rate of appearance of terminal leukemia was directly related to the number of transferred leukemic lymphoblasts. As few as 20 leukemic cells were able to cause a cancer relapse resulting in terminal leukemia 21 days after transplantation in three of five transplanted animals. Our results demonstrate that germ cell transplantation with the presently used techniques is not safe enough for clinical use. Improved methods for purging testicular specimens of cancer cells or totally new approaches with transient xenogenetic host models to detect contamination of malignant cells must be developed before this technique can be offered to patients without fear of disease relapse.     

Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganp^Tg mice). In the GANP−teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganp^Tg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.