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We report a case of intratesticular endometrioid papillary cystadenocarcinoma. A 73-year-old man was admitted for a painless right scrotal swelling. Ultrasonography and computed tomography revealed a large cystic mass in the right testis. Right scrotum puncture revealed xanthochromic fluid with negative cytology. Three months later, follow-up computed tomography showed enlargement of the cystic mass. Right high orchiectomy was performed because a testicular malignancy was suspected. The pathological diagnosis was endometrioid papillary cystadenocarcinoma, and the cells were strongly positive for the estrogen and progesterone receptors. Testicular neoplasms resembling common ovarian-type epithelial tumors are very rare. This is the first report of endometrioid papillary cystadenocarcinoma of the testis.

Context: Leydig cell tumors (LCTs) are the most common non-germ-cell neoplasms of the testis. LCTs are often hormonally active and can result in precocious virilization or in adult feminization. We identified an LCT in an affected individual from a kindred with hereditary leiomyomatosis and renal cell cancer (HLRCC) and a germline fumarate hydratase (FH) mutation (N64T)., Objective: Our objective was to investigate the role of FH mutations in predisposition to LCTs., Design: We tested for pathogenic effects of the N64T mutation and screened an additional 29 unselected adult LCTs for FH alterations. We also tested these LCTs for mutations in two genes, the LH/choriogonadotropin receptor (LHCGR) and the guanine nucleotide-binding protein alpha (GNAS) that had been implicated in LCT tumorigenesis., Results: No mutations were found in GNAS, and one tumor had a LHCGR somatic substitution. In addition to the HLRCC case with the N64T germline FH mutation, we identified one other LCT with a previously unreported FH mutation (M411I). Both LCTs from these patients showed loss of the wild-type FH allele. Immunohistochemical and in situ hybridization analyses demonstrated activation of the hypoxia/angiogenesis pathway not only in the tumors belonging to the FH mutation carriers but also in several other mutation-negative LCTs., Conclusions: Our study shows that some LCTs are caused by FH mutations and represents one of the first reports of germline mutations in any type of adult testicular tumor.

Purpose: The purpose of the study was to define the histological origin of gonadoblastomas, allowing the identification of high-risk patients., Experimental Design: Sixty paraffin-embedded gonadectomy or gonadal biopsy samples of 43 patients with gonadal dysgenesis were selected from our archives. We studied the morphology and immunohistochemical properties of the germ cells in 40 samples without neoplastic transformation and compared these findings with the morphological and immunohistochemical characteristics of 20 samples containing gonadoblastoma/dysgerminoma., Results: The overall incidence of germ cell tumors in our patient series was 35%. In dysgenetic gonads without germ cell neoplasia, besides the presence of areas with testicular and/or ovarian differentiation, areas of undifferentiated gonadal tissue were identified in 13 of 40 samples (32.5%). A subpopulation of germ cells within these undifferentiated areas stained positive for octamer binding transcription factor (OCT)3/4, the stem cell factor receptor, placental-like alkaline phosphatase, and testis-specific protein-Y encoded. Gonadoblastoma germ cells display identical staining results. Moreover, in gonads containing gonadoblastoma, adjacent to this lesion, areas of undifferentiated gonadal tissue with identical immunohistochemical characteristics were identified in 10 of 20 samples (50%). No adjacent tissue was available in five cases, whereas in the five remaining cases, it consisted of streak tissue. In three cases, an accumulation of OCT3/4-positive germ cells in the proximity of the malignant lesions was found, suggesting clonal expansion and final organization into gonadoblastoma nests., Conclusions: Based on these observations, we hypothesize that gonadoblastomas originate from surviving OCT3/4-positive germ cells in areas of undifferentiated gonadal tissue within the dysgenetic gonad. Supportive evidence was obtained that carcinoma in situ arises in regions with testicular differentiation.

Myeloid sarcoma is a neoplasm of immature granulocytes, monocytes, or both involving any extramedullary site. Myeloid sarcoma involving the testis, however, is uncommon and very rarely occurs as an isolated mass. We describe 4 patients with myeloid sarcoma involving the testis, including 2 patients in whom the neoplasm was isolated to the testis (1 unilateral and 1 bilateral). Histologically, 4 neoplasms were poorly differentiated and 1 was blastic. Each neoplasm was shown to be of myeloid lineage, and negative for T- and B-cell specific antigens using immunohistochemical methods. One case was also positive for chloroacetate esterase. In the literature, most cases of myeloid sarcoma involving the testis represent relapse or the initial presentation of acute myeloid leukemia. Including the 2 cases we report here, only 7 cases of myeloid sarcoma isolated to the testis have been reported.

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHS-R), has been primarily linked to the central neuroendocrine regulation of GH secretion and food intake, although additional peripheral actions of ghrelin have also been reported. In this context, the expression of ghrelin and its cognate receptor has been recently demonstrated in rat testis, suggesting a role for this molecule in the direct control of male gonadal function. However, whether this signaling system is present in human testis remains largely unexplored. In this study we report the expression and cellular location of ghrelin and its functional receptor, the type 1a GHS-R, in adult human testis. In addition, evaluation of ghrelin and GHS-R1a immunoreactivity in testicular tumors and dysgenetic tissue is presented. The expression of the mRNAs encoding ghrelin and GHS-R1a was demonstrated in human testis specimens by RT-PCR, followed by direct sequencing. In normal testis, ghrelin immunostaining was demonstrated in interstitial Leydig cells and, at lower intensity, in Sertoli cells within the seminiferous tubules. In contrast, ghrelin was not detected in germ cells at any stage of spermatogenesis. The cognate ghrelin receptor showed a wider pattern of cellular distribution, with detectable GHS-R1a protein in germ cells, mainly in pachytene spermatocytes, as well as in somatic Sertoli and Leydig cells. Ghrelin immunoreactivity was absent in poorly differentiated Leydig cell tumor, which retained the expression of GHS-R1a peptide. In contrast, highly differentiated Leydig cell tumors expressed both the ligand and the receptor. The expression of ghrelin and GHS-R1a was also detected in dysgenetic Sertoli cell-only seminiferous tubules, whereas germ cell tumors (seminoma and embryonal carcinoma) were negative for ghrelin and were weakly positive for GHS-R1a. In conclusion, our results demonstrate that ghrelin and the type 1a GHS-R are expressed in adult human testis and testicular tumors. Overall, the expression of ghrelin and its functional receptor in human and rat testis, with roughly similar patterns of cellular distribution, is highly suggestive of a conserved role for this newly discovered molecule in the regulation of mammalian testicular function.

We studied 10 cases of primary pure testicular neuroendocrine carcinoma. Patients were between 16 and 48 years old and had testicular swelling with pain or a painless testicular mass and no history of neuroendocrine carcinoma or other malignant neoplasm. All underwent orchiectomy. The tumors were low (n = 9) and intermediate (n = 1) grades with a variegated histologic appearance characterized by a nesting pattern, cords of neoplastic cells with rosettes, or sheets of neoplastic cells. Mitotic activity was lacking in 9 cases. In 1 case, mitotic figures ranged from 7 to 8 per 10 high-power fields, and cellular atypia and comedo-like necrosis were present. Immunohistochemical studies using a keratin cocktail, chromogranin, synaptophysin, epidermal growth factor, p53, placental-like alkaline phosphatase, and CD117 (c-kit) were performed in all cases. Keratin, chromogranin, and synaptophysin were positive in all tumors. Clinical follow-up information was obtained for 6 patients (range, 12-60 months): 5 with low-grade tumors were alive 24 to 60 months after diagnosis; 1 with an intermediate-grade tumor died of tumor 12 months after initial diagnosis. The behavior of these tumors, while in the testicular region, correlates well with the histologic grade. We propose replacing the term testicular carcinoid with neuroendocrine carcinoma, which better reflects the nature of these neoplasms.

We assessed the usefulness of fine-needle aspiration biopsy (FNAB) in the diagnosis of mediastinal germ cell tumors (GCTs). In the archives of 3 pathology departments, we found records of 7 patients with mediastinal GCTs who underwent mediastinal FNAB as part of the diagnostic workup. The FNAB smears, results of the immunocytochemical analysis, the corresponding histologic findings, and the clinical charts were reviewed. All patients were men (age range, 24-44 years; mean, 32 years). One patient had a history of testicular mixed GCT 10 years earlier. The 6 primary mediastinal GCTs consisted of 3 seminomas and 3 yolk sac tumors. Based on the cytologic features and immunocytochemicalfindings, a cytologic diagnosis of GCT was made in 5 cases, including the case of metastatic GCT In 2 cases, the differential diagnosis was between poorly differentiated carcinoma and GCT Results of ancillary studies were noncontributory in 1 case, and the aspirate of the second case demonstrated extensive necrosis. Our findings demonstrate that a diagnosis of mediastinal GCT, primary or secondary, can be established with a high degree of accuracy on the basis of FNAB. Immunocytochemical analysis helps confirm the diagnosis.

Objective: Inhibin and activin are proteins produced by ovarian granulosa cells and testicular Sertoli cells and are members of the transforming growth factor-beta superfamily. Since increased circulating levels of immunoreactive inhibin were detected in women with malignant ovarian tumors, they were proposed as tumor markers for ovarian carcinoma. Immunohistochemical studies later confirmed the presence of inhibin and activin subunits in granulosa cell tumors and epithelial ovarian cancer, as well as in Sertoli and Leydig cell testicular cancer. However, there is discrepant information on the detection of inhibin and activin in malignant germ cell tumors (MGCT). The aim of the present study was to evaluate the immunohistochemical expression of the inhibin/activin alpha, betaA and betaB subunits in ovarian and testicular MGCT specimens using polyclonal antisera., Methods: The ovarian tissue samples were composed of 19 MGCT, including dysgerminoma (n=18) and yolk sac tumor (n=1). The testis specimens included classic seminomas (n=20), embryonal carcinomas (n=7), choriocarcinomas (n=2), and yolk sac tumor (n=1)., Results: Ovarian and testicular malignant germ cell tumors expressed positive staining for inhibin/activin alpha, betaA and betaB subunits, with some variations between and within individual tumors: while ovarian dysgerminomas were diffusely positive for alpha, betaA and betaB, testicular tumors expressed alpha and betaB subunits, whereas betaA staining was weak., Conclusions: The present results show positive staining for inhibin/activin subunits in ovarian and testicular MGCT, suggesting a possible role in tumorigenesis with the resultant clinical implication.

The immunohistochemical reaction to oncostatin M (OSM) was studied in normal human testes at different ages (fetuses, newborns, children, pubertal boys, adults, and elderly men), as well as in several testicular disorders including carcinoma-in-situ cells (CIS), germ cell tumors, benign functioning Leydig cell tumor, androgen insensitivity syndrome, Klinefelter's syndrome, and cryptorchidism. Positive OSM immunostained Sertoli cells were only observed in fetuses. In normal testes, intense OSM immunoreaction was found in the Leydig cells of fetuses, newborns, and adults. Leydig cell immunoreaction was weak in elderly men and absent in children and pubertal boys. In some testicular disorders (Leydig cell tumor, cryptorchidism, and CIS), Leydig cell immunoreaction was as intense as in normal adult testes. This immunoreaction was heterogeneous in androgen insensitivity syndrome and was absent in Klinefelter's syndrome and intratubular seminoma. No recognizable Leydig cells were observed in the other testicular tumors. The findings of our study suggest that, in humans, the down-regulation of OSM immunoexpression in Sertoli cells occurs early, and that OSM immunoreaction in the Leydig cells is associated with functionally active and differentiated Leydig cells.

We previously demonstrated that the mitochondrial peripheral-type benzodiazepine receptor (PBR) is coupled to hormone-activated steroidogenesis by regulating the intramitochondrial cholesterol transport, the rate-determining step of steroid biosynthesis. In the present study we examined whether PBR is the site of hormone action using the hCG-responsive MA-10 mouse Leydig tumor cell line as a model system. Within 15 sec of the addition of hCG to Leydig cells a 3-fold cAMP-dependent increase in PBR binding was observed. This rapid increase returned to basal levels within 60 sec. No effect was observed after 1 min in the continued presence of hCG. Scatchard analysis revealed that in addition to the known high affinity (5.0 nM) benzodiazepine-binding site, a second, hormone-induced, higher affinity (0.2 nM) benzodiazepine-binding site appeared. We then examined whether in such a short time frame steroid synthesis occurs. Fifteen-second incubation of MA-10 cells with the inhibitor of cholesterol metabolism aminoglutethimide together with hCG also resulted in an increased rate of pregnenolone formation by their isolated mitochondria that were washed and incubated in aminoglutethimide-free buffer. The dose response of benzodiazepine binding to hCG closely parallels the increase in steroid formation by the mitochondria of stimulated cells. Addition of the selective inhibitor of cAMP-dependent protein kinase, H-89, completely blocked hormone-induced PBR binding and steroid formation, whereas addition of the inactive analog H-85 was without any effect. The addition of flunitrazepam, a benzodiazepine previously shown to inhibit the trophic hormone action on steroidogenesis, completely abolished the hCG-induced rapid stimulation of steroid synthesis. These results demonstrate that in MA-10 cells, the most rapid effect described thus far of hCG and cAMP, is the transient induction of a higher affinity benzodiazepine-binding site, which occurs concomitantly with an increase in the rate of steroid formation. This, in turn, suggests that these hormones alter PBR to activate cholesterol delivery to the inner mitochondrial membrane and subsequent steroid formation.

In biological fluids, hCG and its free alpha- (hCG alpha) and beta-subunits (hCG beta), occur in multiple forms. These various forms differ at the molecular level primarily in glycosylation, but also differ in protein backbone modifications corresponding to the urinary low molecular weight fragment of the hCG beta-subunit (beta-core fragment). This microheterogeneous nature can be demonstrated by isoelectric focusing in which variants are separated into bands with different isoelectric points (pI). To determine whether such isoelectric variants differ in antigenicity and consequently might escape immunoassay detection due to overspecificity of monoclonal antibodies (MCA), urinary pregnancy hCG (NIH, CR123) and tumor hCG preparations, such as a tumor-specific acidic variant of hCG (hCGav) and the hCG beta-core fragment, were separated by isoelectric focusing in the absence or presence of 8 M urea, or by sodium docedyl sulfate-polyacrylamide gel electrophoresis and enzymatically immunostained using an MCA panel directed against 17 different hCG epitopes. MCA against 14 different epitopes accessible on holo-hCG recognized all pI variants of pregnancy holo-hCG or tumor-derived hCGav, as was true for the three MCA recognizing epitopes hidden on holo-hCG but accessible on the free subunits after hCG dissociation by urea. We conclude that each individual pI-isoform of holo-hCG and its free subunits expresses the entire set of epitopes recognized by our MCA panel. The carbohydrate moieties that form a biochemical basis for hCG heterogeneity seem to be neither of major antigenic relevance, nor are they structurally related to any particular epitope. Thus, various glycosylation forms of hCG, hCG alpha, hCG beta, and hCG beta-core in normal as well as in pathological samples should safely be detectable and measureable by immunoassays employing MCA with appropriate subunit specificity.

A case of malignant sex-cord stromal tumor (gonadal stromal tumor) in the left testis of a 42-year-old man with no sign of hormonal abnormality is reported. Histologically, a large portion of the tumor was composed of solid nests of round cells with a moderate amount of eosinophilic cytoplasm, but some areas contained small spaces somewhat resembling Call-Exner bodies and/or the follicular structures of granulosa cell tumors. In general, however, the tumor cells did not show a clear-cut differentiation into either Sertoli cells or granulosa cells. The tumor, because of its high mitotic rate, lymphatic, venous and capsular invasion, and metastasis to the para-aortic lymph nodes, was considered to be malignant. We simply designated this tumor, therefore, as a malignant sex-cord stromal tumor. An ultrastructural study revealed unique intracytoplasmic bodies composed of multiple stacks of tubular structures which superficially mimicked annulate lamellae. No lipid droplets, Reinke's crystalloids or Charcot-Böttcher filaments were seen. The rarity of the tumor and the presence of unusual substructures prompted us to report the case.

Sertoli cell intracellular protein 1 (SCc1) and 2 (SCc2) are polypeptides found in rat Sertoli cell cultures incubated with either FSH or (Bu)2cAMP. They were first identified in [35S]methionine-labeled Sertoli cell lysates using two-dimensional gel electrophoresis. Here we extend these observations by showing that SCc1 and SCc2 are present in rat seminiferous tubules, ovaries, and granulosa cells incubated with either FSH or (Bu)2cAMP and in testicular peritubular cells incubated with (Bu)2cAMP. Peritubular cells do not, however, respond to FSH with the production of SCc1 and SCc2. Peptide mapping with N-chlorosuccinimide revealed that SCc1 and SCc2 have similar cleavage patterns, suggesting a common primary amino acid sequence that is modified posttranslationally. Metabolic labeling with [32P]orthophosphate provided direct evidence that SCc1 and SCc2 are phosphoproteins. A shift in mobility of SCc1 and SCc2 toward the basic region of the gel to positions designated SCc1' and SCc2' occurred when cell lysates were treated with alkaline phosphatase before electrophoresis, providing additional evidence that SCc1 and SCc2 are phosphoproteins. SCc1 and SCc2 are also shown to be mitochondrially-associated in the Sertoli cell. Peptide maps of SCc1, SCc2, SCc1', and SCc2' obtained by treatment with alpha-chymotrypsin, are identical to proteolytic maps of proteins pp30', p30, and pp30 from adrenocortical cells. SCc1, SCc2, SCc1', and SCc2' are homologous with regard to their regulated expression, electrophoretic mobility, and mitochondrial localization to the adrenal proteins pp30' and pp30 as well as a series of 30 kilodalton proteins from MA-10 Leydig tumor cells. Both the adrenal cell proteins and the Leydig tumor cell proteins are thought to participate in cholesterol transport to the inner mitochondrial membrane, providing substrate for the cholesterol side-chain cleavage enzyme complex, an activity which the Sertoli cell does not perform, suggesting that alternative functions must be sought for SCc1 and SCc2 in Sertoli cells.

Recent ultrastructural, cytogenetic, and ploidy analyses indicate that seminoma acts as a precursor from which other forms of testicular germ cell tumor may originate. Ten cases of primary or metastatic testicular germ cell tumors were investigated that showed possible transformation of seminoma to yolk sac tumor. Such transformation was identified in six cases in which foci of abrupt change from seminoma to various patterns of yolk sac tumor occurred, often at the periphery of otherwise pure lobules of seminoma. Immunostains for cytokeratins, placental-like alkaline phosphatase, and alpha-fetoprotein demonstrated the expected changes in reactivity at the foci of such transformation. Four additional cases were regarded as either seminomas with artifactual microcystic change or the close association of seminoma and yolk sac tumor but lacking evidence for transformation. These data support the theory that seminoma is not an "endpoint" neoplasm but may serve a precursor role in the progression to nonseminomatous germ cell tumors.

Monoclonal antibodies (MAb; DMAb, monoclonal antibodies derived at Duke Medical Center) directed against the oncofetally expressed lactotetraosyl gangliosides 3'-isoLM1 (IV3NeuAc-LcOse4Cer) and 3',6'-isoLD1 (IV3NeuAc,III6NeuAc-LcOse4Cer) were produced and their reactivity spectra compared to that of the alpha-3'-isoLM1 MAb SL-50. The IgM MAb SL-50 defines the epitope NeuAc (or NeuGc)alpha 2-3Gal beta 1-3GlcNAc, the terminal sequence of both gangliosides. SL-50 requires an unsubstituted GlcNAc residue; IgM DMAb-14 will accept the alpha 2-6 linked sialic acid to GlcNAc found in 3',6'-isoLD1. Immunohistochemical localization of 3'-isoLM1 was performed on 31 biopsy specimens of human gliomas; 15 (48%) expressed 3'-isoLM1 as defined by binding of MAb SL-50. Staining of small anaplastic cells, giant cells, and the glial component of gliosarcomas was observed. Neoplastic gemistocytes, when present, showed particularly intense staining. The 3'-isoLM1 and 3',6'-isoLD1 distribution in cultured cell lines and derived xenografts of primary tumors of the human central nervous system and of embryonal or neuroectodermal tumor derivation was determined. Six of 29 cell lines expressed 3'-isoLM1: 2/16 gliomas, 3/3 teratomas, 1/1 pancreatic adenocarcinoma. No cell line expressed detectable 3',6'-isoLD1 by immunostain analysis of ganglioside extracts. The 3'-iso-LM1-positive cell lines expressed it in xenograft form; in five xenografts, the corresponding cell lines of which were 3'-isoLM1-negative, it was a proportion of the monosialoganglioside fraction. 3',6'-isoLD1 was detected in two xenografts, D-54 MG (glioma) and PA-1 (teratoma). The demonstration of 3'-isoLM1 in gliomas in in vivo forms and the relatively infrequent expression by derived cultured cells suggest that ganglioside expression is modified by environmental forces. Expression of 3'-isoLM1 and 3',6'-isoLD1 in fetal and neonatal brain, in intense reactive astrocytosis such as polyunsaturated fatty acid lipidosis, and in primary neoplasms of the central nervous system suggests their role in cell-cell attachment during development, migration, and neoplastic transformation.

PTH-like peptide (PLP) is produced by tumors commonly associated with hypercalcemia as well as nonneoplastic tissues and several endocrine glands and tumors. To characterize the distribution of PLP in human endocrine tissues and tumors, we localized PLP in formalin-fixed paraffin-embedded material using the avidin-biotin-peroxidase technique with polyclonal antisera. Among peptide hormone-producing tissues, PLP was identified in nontumorous adenohypophysis and pituitary adenomas; medullary thyroid carcinomas; normal, hyperplastic, and adenomatous parathyroids; adrenal medulla and pheochromocytomas; normal pancreatic islets; and endocrine tumors of pancreas, gut, and lung, including small cell carcinomas. In other endocrine tissues PLP was identified in nontumorous thyroid follicular epithelium, colloid nodules, and follicular neoplasms; normal adrenal cortex, adrenocortical adenomas, and carcinomas; nontumorous testicular Leydig cells; normal ovarian granulosa and thecal cells; an ovarian thecoma and a granulosa cell tumor; placental trophoblast; and decidua. These results demonstrate that PLP is localized in many normal and neoplastic endocrine cells, including those not known to influence extracellular calcium homeostasis. The presence of PLP in a variety of endocrine tissues suggests that it may play a local physiological role in the growth or function of endocrine cells.