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We previously demonstrated a genetic evidence of the progression from seminoma to embryonal carcinoma in mixed testicular germ cell tumors (TGCTs). This process, the "reprogramming" of seminoma cells, is crucial for pathological tumorigenesis and should be kept in mind while designing clinical therapeutic strategies. We hypothesized that a comparison between pure-type seminomas and seminoma components in mixed tumors (mixed-type seminomas) could reveal early changes in the reprogramming process. In the present study, we performed gene expression microarray analysis of six pure-type and six mixed-type seminomas. Hierarchical clustering analysis properly grouped each type of seminomas into a separated cluster. Supervised analysis between pure-type and mixed-type seminomas revealed 154 significantly dysregulated genes (Storey-adjusted q < 0.05). The genes with the highest overexpression in mixed-type seminomas compared with the pure-type seminomas included MT1 isoforms, PRSS8, TSC22D1, and SLC39A4; downregulated genes included DEFB123, LMTK2, and MYRF. Functional annotation analysis of the differentially expressed genes revealed that the top-ranked functional categories were related to cellular zinc metabolism and consisted of MT1 isoforms and SLC39A4, the results of which were validated using quantitative polymerase chain reaction and immunohistochemical analysis. In conclusion, this research provides further evidence that pure and mixed types of seminomas are molecularly different, which may contribute to elucidate the reprogramming mechanism in the progression of TGCTs., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lymphovascular invasion (LVI) is a relevant prognostic factor in germ cell tumors of the testis (GCTT) and it has been included in the AJCC staging system. Nevertheless, its histological assessment is challenging, with low/moderate interobserver agreement also among expert uropathologists. Few studies focused on the potential role of immunohistochemistry to solve this critical issue; as result, in current guidelines there is no indication for additional staining to detect this histological feature. In the present study, we investigated the detection of LVI invasion in a small cohort of GCTT with double staining for OCT4/CD34. Although our results need to be validated in larger case series with follow-up data, they suggest as OCT4/CD34 could be a useful tool for the histological assessment of these tumors, helping to identify some histological mimickers of LVI and modifying the pT/stage in a significant percentage of patients., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Background: Better biomarkers for assessing risk of relapse in stage I testicular germ cell tumor patients are needed, to complement classical histopathological variables. We aimed to assess the prognostic value of previously suggested biomarkers, related to proliferation (MIB-1 and TEX19) and to immune microenvironment (CXCL12, CXCR4, beta-catenin and MECA-79) in a surveillance cohort of stage I testicular germ cell tumor patients., Methods: A total of 70 patients were included. Survival analyses were performed, including Cox regression models., Results: Patients with vascular invasion and elevated human chorionic gonadotropin levels showed significantly poorer relapse-free survival in multivariable analysis (hazard ratio = 2.820, 95% confidence interval 1.257-6.328; hazard ratio = 3.025, 95% confidence interval 1.345-6.808). Patients with no vascular invasion but with MIB-1 staining in > 50% tumor cells showed significantly shorter relapse-free survival (p = 0.042). TEX19 nuclear immunoexpression was confirmed in spermatogonial cells, and weak cytoplasmic immunoexpression was depicted in 15/70 tumors, not significantly impacting survival. CXCL12 immunoexpression in tumor cells did not associate with relapse, but non-seminoma patients exhibiting vascular invasion and CXCL12-positive stromal/inflammatory cells showed significantly improved relapse-free survival (p = 0.015). Exclusively nuclear immunoexpression of CXCR4 associated with better relapse-free survival (p = 0.032), but not after adjusting for vascular invasion. Patients with higher beta-catenin scores showed a tendency for poorer relapse-free survival (p = 0.056). MECA-79 immunoexpression was absent., Conclusions: The informative protein biomarkers (i.e., MIB-1, CXCL12, beta-catenin, and possibly CXCR4) may prove useful for risk-stratifying patients if validated in larger, multicentric and well-defined studies. Currently, classical histopathological features of testicular germ cell tumors remain key for relapse prediction.

In the present study, we investigated protein expression of the transcription factors mammalian doublesex and mab-3 related transcription factor 1 (DMRT1), basic helix-loop-helix transcription factor-like 5 (TCLF5), and octamer-binding transcription factor 4 (OCT4) in normal human spermatogenesis, testicular mixed germ cell-sex cord stromal tumor (MGC-SCST), spermatocytic tumor, and seminoma. In normal human spermatogenesis, DMRT1 is expressed in the nuclei of spermatogonia but not in those of more mature germ cells. By way of contrast, TCLF5 is expressed in the nuclei of some clusters of primary spermatocytes that have entered meiosis 1, in secondary spermatocytes, and in round (early) spermatids in the seminiferous tubules of adults during the reproductive years. OCT4 is expressed in primordial germ cells but not in the seminiferous tubules of the normal adult testis during the reproductive years. DMRT1 is expressed in the germ cells of both testicular MGC-SCST and spermatocytic tumor, whereas TCLF5 is not expressed in either neoplasm. These low-grade neoplasms, however, differ histologically in that all the germ cell nuclei of testicular MGC-SCST resemble spermatogonia, whereas in spermatocytic tumor, the nuclei of the medium-sized and large cells resemble those of primary spermatocytes. Both neoplasms lack expression of OCT4. By way of contrast, in seminoma, a fully malignant testicular germ cell tumor, the germ cell nuclei express OCT4 but do not express either DMRT1 or TCLF5., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Histologically pure seminoma with elevated alpha-fetoprotein (AFP), so-called AFP-positive seminoma, is rare. It is recommended that patients with AFP-positive seminoma be managed as non-seminoma, but the clinical features and prognosis of this disease are not fully understood. In this study, we retrospectively analyzed 6 cases of metastatic AFP-positive seminoma at Tsukuba University Hospital (TUH). AFP was elevated before induction chemotherapy in 4 patients with an average of 1,372 ng/ml. In the remaining 2 patients, AFP became elevated during or after induction chemotherapy. In all 4 patients examined, AFPL3% was abnormally increased. As induction chemotherapy, all patients received bleomycin, etoposide and cisplatin (BEP), which was then followed by etoposide, ifosfamide and cisplatin (VIP) in 3 patients. After or during induction chemotherapy, 3 patients suffered from disease progression accompanied by AFP elevation. All 3 were treated by salvage chemotherapy and surgery. Four patients underwent retroperitoneal lymph node dissection (RPLND) after induction chemotherapy ; the pathological findings were necrosis in 3 patients, and viable nonseminomatous cancer in 1 patient. Furthermore, RPLND was performed as salvage surgery in 3 patients ; the pathological findings were necrosis, viable nonseminomatous cancer and teratoma with malignant transformation, respectively. The 5-year progression-free survival rate of the 6 patients was 50%, which is somewhat inferior to that of poor-prognosis non-seminoma patients treated at TUH. One patient ultimately died of cancer, and the remaining 5 are in remission with a median follow-up of 58 months. The present study demonstrates that AFP-positive seminoma patients have a higher risk of relapse compared to non-seminoma patients.

Testicular germ cell tumor (TGCT) is the most common malignancy of young men. Most patients are completely cured, which distinguishes these from most other malignancies. Orchiectomy specimens (n=76) were evaluated using high-resolution (single-cell discriminative) telomere-specific fluorescence in situ hybridization (FISH) with simultaneous Oct4 immunofluorescence to describe telomere length phenotype in TGCT neoplastic cells. For the first time, the TGCT precursor lesion, germ cell neoplasia in situ (GCNIS) is also evaluated in depth. The intensity of the signals from cancerous cells was compared to the same patient's reference cells-namely, healthy germ cells (defined as "medium" length) and interstitial/somatic cells (defined as "short" telomere length). We observed short telomeres in most GCNIS and pure seminomas (P=.006 and P=.0005, respectively). In contrast, nonseminomas displayed longer telomeres. Lesion-specific telomere lengths were documented in mixed tumor cases. Embryonal carcinoma (EC) demonstrated the longest telomeres. A fraction of EC displays the telomerase-independent alternative lengthening of telomeres (ALT) phenotype (24% of cases). Loss of ATRX or DAXX nuclear expression was strongly associated with ALT; however, nuclear expression of both proteins was retained in half of ALT-positive ECs. The particular distribution of telomere lengths among TGCT and GCNIS precursors implicate telomeres anomalies in pathogenesis. These results may advise management decisions as well., (Copyright © 2016. Published by Elsevier Inc.)

We previously identified TDRG1 (testis developmental related gene 1), a novel gene with exclusive expression in testis, promoted the proliferation and progression of cultured human seminoma cells through PI3K/Akt/mTOR signaling. As increasing evidence reveal that aberrant activation of this signaling is involved in cisplatin resistance. Then, in this study, we further explored whether TDRG1 regulated the chemosensitivity of seminoma TCam-2 cells to cisplatin. Our researches showed TDRG1 could regulate the viability of TCam-2 cells following cisplatin treatment in vitro through control of both cell apoptosis and cell cycle. Mechanistically, we observed TDRG1 positively regulated the expression levels of the key elements in PI3K/Akt/mTOR pathway including p-PI3K, p-Akt and p-mTOR and also affected the translocation of nuclear p-Akt in TCam-2 cells during cisplatin treatment. Meanwhile, the levels of Bad, cytochrome c, caspase-9 ratio (activated/total), caspase-3 ratio (activated/total) and cleaved-PARP were negatively modulated by TDRG1, which meant the involvement of mitochondria-mediated apoptotic pathway. Furthermore, we found the effect of TDRG1 knockdown or TDRG1 overexpression could be reversed by IGF-1, a PI3K signaling activator, or LY294002, a inhibitor of this pathway, respectively. Similar effects of TDRG1 on cisplatin chemosensitivity and associated molecular mechanism were also confirmed in vivo by employing xenograft assays. In addition, the positive correlation between TDRG1 and p-PI3K, or p-Akt, was found in tumor tissues from seminoma patients. In conclusion, we uncover that TDRG1 regulates chemosensitivity of TCam-2 cells to cisplatin through PI3K/Akt/mTOR signaling and mitochondria-mediated apoptotic pathway both in vitro and in vivo.

Aims: First, to determine the frequency of intravascular granulomas (IVGs) in seminomas and assess for the presence of entrapped seminoma cells. Second, to identify the relationship of this unusual form of vascular space invasion with tumour relapse and/or dissemination., Methods: 86 cases of seminoma were reviewed to identify IVGs. Immunostaining for OCT3/4 and CD68 was performed. Pathological stage, presence of conventional vascular and rete testis invasion, parenchymal granulomas and follow-up were recorded. Multivariable analysis incorporating tumour size, vascular invasion (conventional granulomas and IVGs) and rete testis invasion was performed., Results: IVGs were identified in 13 cases (13/86). CD68 confirmed histiocytes in all cases. OCT3/4 identified tumour cells in 9/13 seminomas. 27 patients had disease progression with either dissemination at presentation (n=11) or relapse (n=16). Of these 27 patients, 8 had IVG (29.6%). By comparison, 6 of 57 clinical stage 1 seminomas that did not relapse had IVG (10.53%). Multivariable analysis revealed that no single parameter was statistically significant at predicting tumour relapse and/or dissemination (size: HR 1.65; CI 0.71 to 3.82, p=0.24, rete testis invasion: HR 1.04; CI 0.48 to 2.26, p=0.92, lymphovascular space invasion/IVG: HR 1.62; CI 0.65 to 4.01, p=0.30)., Conclusions: IVGs may represent a previously unrecognised form of vascular space invasion in seminomas. Studies on larger cohorts are needed to demonstrate its clinical value., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Primary mediastinal seminomas are unusual tumors that can present in a pure form or as part of a mixed germ cell tumor. Contrary to testicular seminomas, little is known about the expression of novel immunohistochemical markers in mediastinal seminomas. This study investigates the immunohistochemical features of these tumors with a focus on novel markers. Thirty-two cases of primary mediastinal seminomas were reviewed; and representative whole-tissue sections were selected for immunohistochemical studies using antibodies directed against high molecular weight cytokeratin 5/6 (CK5/6), low molecular weight cytokeratin (CAM5.2), octamer-binding transcription factor 3/4 (OCT3/4), spalt-like transcription factor 4 (SALL4), GATA binding protein 3 (GATA-3), sry-related HMG box 2 (SOX2), SOX17, human T cell leukemia/lymphoma 1 (TCL1), glypican 3, melanoma associated antigen C2 (MAGEC2), and paired box gene 8 (Pax8). The percentage of positive tumor cells as well as the intensity of staining was evaluated and scored. Thirty-one cases (97%) expressed SOX17, whereas 29 cases (91%) were positive for OCT3/4 and SALL4, respectively. Twenty-eight cases (88%) expressed MAGEC2 and CAM5.2, respectively. Two cases (6%) were positive for Pax8, and a single case (3%) was positive for TCL1. None of the cases stained with CK5/6, GATA-3, SOX2, or glypican 3. Similar to testicular seminomas, mediastinal seminomas show consistent expression of OCT3/4, SALL4, SOX17, and MAGEC2 and are negative for SOX2, glypican 3, GATA-3, and CK5/6. Pax8 positivity is only inconsistently identified in mediastinal seminomas. Contrary to their testicular counterparts, mediastinal tumors show diffuse expression of low-molecular-weight cytokeratin in up to 90% of cases and are commonly negative for TCL1. Although there is some immunohistochemical overlap between testicular and mediastinal seminomas, considerable differences also exist and should be acknowledged when dealing with these tumors., (Copyright © 2015 Elsevier Inc. All rights reserved.)

All malignant testicular germ cell tumors (TGCT) of adult men are preceded by an in situ stage (CIS) of protracted evolution. The adult CIS is well characterized, but there is debate on the phenotype of infantile CIS, its distinction from delayed maturation of germ cells and prognostic potential. A large series of 43 patients with Disorders of Sex Development (DSD) and dysgenetic testes (90% ranging from neonates to 12 years, mean age 4.7 years), was studied by quantifying dysgenetic features, degree of germ cell abnormalities/atypia (GCA), expression of OCT 3/4 (a pluripotency-undifferentiation marker), germ cell ploidy and evolution to CIS and invasive TGCT. Findings were compared with those of normal testes. The type of gonads present defined three groups of patients: bilateral testes (BT-DSD, n = 21), one testis and one streak gonad (CT-DSD, C for combined, n = 13), and ovarian-testicular combinations (OT-DSD, n = 9). There were 5 boys with infantile CIS, bilateral in 3 (total of 8 infantile CIS) and two patients with adult CIS, bilateral in one (total of 3 adult CIS). Two patients had bilateral seminomas one at 12-17 and the other at 23 years. Histological dysgenesis was significantly higher in CT-DSD (p < 0.05), that had only 1 CIS. The highest frequency of GCA was in BT-DSD (p < 0.05), which coincided with a total of 11CIS + Seminomas. In all patients, aneuploidy was significantly higher (63%) than diploidy (p < 0.02), and GCA were more frequent in aneuploid than in diploid samples (p < 0.02). All CIS and TGCT were OCT 3/4 positive. Finally, there was a significant association between the triad Aneuploidy + GCA + OCT 3/4 positivity and the incidence of CIS (Fisher Exact test p < 0.002, relative risk 7.0). The degree of testicular dysgenesis (derived from abnormal organization of Sertoli cells in fetal testicular cords) is inversely related to the incidence of CIS. Our data demonstrate that the combined use of OCT 3/4 expression, quantification of germ cell abnormalities-atypia and ploidy in dysgenetic testes can satisfactorily identify infantile CIS with high risk of malignant evolution and set it aside from delayed germ cell maturation with lower or nil neoplastic potential., (© 2014 American Society of Andrology and European Academy of Andrology.)

TCam-2 cells are the main in vitro model for investigations into seminomatous tumors. However, despite their widespread use, questions remain regarding the cells' homogeneity and consequently how representative they are of seminomas. We assess the TCam-2 cell line using routine and novel authentication methods to determine its homogeneity, identify any cellular sub-populations and resolve whether any changes could be due to generational differentiation. TCam-2, embryonal carcinoma cells (2102EP) and breast cancer cell (MCF7) lines were assessed using qRT-PCR, immunocytochemistry, flow cytometry and short tandem repeat analyses. Raman maps of individual cells (minimum of 10) and single scan spectra from 200 cells per culture were obtained. TCam-2s displayed the characteristic marker gene expression pattern for seminoma, were uniform in size and granularity and short tandem repeat analysis showed no contamination. However, based only on physical parameters, flowcytometry was unable to differentiate between TCam-2 and 2102EPs. Raman maps of TCam-2s comprised three equally distributed, distinct spectral patterns displaying large intercellular single spectral variation. All other cells showed little variation. Principal component, cluster and local spectral angle analyses indicated that the TCam-2s contained two different types of cells, one of which comprised two subgroups and was similar to some 2102EP cells. Protein expression corroborated the presence of different cells and generational differences. The detailed characterization provided by the Raman spectra, augmented by the routine methods, provide substantiation to the long-held suspicion that TCam-2 are not homogeneous but comprise differing cell populations, one of which may be embryonal carcinoma in origin.

Background: Spermatocytic seminoma is a rare testicular germ cell tumor of old men. Accounting for 1-4% of all seminomas, spermatocytic seminomas have distinct pathogenesis, histological features, immunohistochemical profile and comparatively benign clinical behavior which distinguishes them from other germ cell tumors, especially classic seminoma., Aims: The purposes of our study were to assess the patient demographics, pathological features and to evaluate the utility of CD 117 immunostain along with other immunohistochemical stains in distinguishing Spermatocytic seminomas from classic seminomas., Material and Methods: All spermatocytic seminomas patients diagnosed during 1992 to 2013 at Section of Histopathology, Department of Pathology and Microbiology, Aga Khan University hospital were included. Patient characteristics, histological details and follow-up data of few patients were available. CD 117 expression was determined by immunohistochemistry., Results: Total 16 cases of Spermatocytic seminomas were reviewed. Median age was 60 years and average tumor size was 10.4 cms. Microscopically, all of the 16 cases showed presence of edema and absence of lymphocytic infiltrate and intratubular germ cell neoplasia. Cytoplasmic glycogen was negative in all 13 cases, PLAP immunostain was negative in all 12 cases, while CD 117 was positive in all 8 cases, where applied., Conclusion: CD 117 is of limited utility in differentiating the spermatocytic seminoma from classic seminoma as it is expressed in significant number of spermatocytic seminomas. However, different histological features, PAS special stain and PLAP immunostain are significantly helpful in distinguishing these two entities.

Testicular spermatocytic seminoma (SS) is a rare tumor type predominantly found in elderly men. It is thought to originate from spermatogonia and shows cytological and genetic heterogeneity. In this study, we performed for the first time a comprehensive analysis of epigenetic modifications in a series of 36 SS samples. We assessed by immunohistochemistry tumor DNA methylation levels, the expression of methyltransferases DNMT3A, DNMT3B and DNMT3L as well as levels of histone modifications H3K9me2, H3K27me3, H3K4me1, H3K4me2/3, H3K9ac, and H2A.Z. We did not identify any epigenetic marks that matched the pattern of the supposed cell-of-origin, the spermatogonia, and found no correlation between specific marks and the size of the SS cells. The emerging epigenetic picture of SS is a heterogeneous "salt-and-pepper"-like pattern, with neighboring cells displaying very variable levels of epigenetic marks. We conclude that SS cells display apparent epigenetic heterogeneity and instability, with loss of the organized manner typical for normal germ cell maturation in the adult testis, likely due to the lack of regulatory signals from the absent somatic cell niche., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Podoplanin, a 38-kd membrane glycoprotein of podocytes, is the human homologue of mouse protein, aggrus, a 44-kd sialoglycoprotein; the latter has recently been reported in testicular seminomas but not in embryonal carcinomas, suggesting that it may be a specific marker for seminomas. The physiologic role of podoplanin has yet to be determined and its function in tumors is not well characterized. In this study we investigate the utility of podoplanin expression in the diagnosis of testicular germ cell tumors. Sixty-eight cases of testicular mixed germ cell tumors were analyzed using a polyclonal antibody and compared to the results for OCT3/4. Stained sections were graded semiquantitatively as follows: negative (no expression), 1+ (mild intensity), 2+ (moderate intensity), 3+ (intense staining). Diffuse cytoplasmic expression of podoplanin with membrane accentuation was seen in the seminoma component of all cases. Lymphocytes and interstitial cells were negative. In mixed germ cell tumors, podoplanin identified small clusters of seminoma cells lying adjacent to nonseminomatous components. Focal staining was present in one third of cases of choriocarcinoma. There was no significant staining of any nontrophoblastic, nonseminomatous elements. In contrast to OCT3/4, podoplanin does not stain embryonal carcinoma. The availability of a marker for seminoma will enable better recognition and distinction from embryonal carcinoma, particularly in nongonadal sites. The identification by podoplanin of seminoma cells lying adjacent to foci of embryonal carcinoma gives credence to the hypothesis that the latter arise from seminomas as part of a process of "differentiation.", (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Human seminoma is classified as classical seminoma (SE) and spermatocytic seminoma (SS). Human SE is known to be more malignant and metastasizing more frequently than SS. Tumor angiogenesis is highly related with tumor progression and metastasis, with microvessel density (MVD) being an important parameter of metastatic potential. Canine seminoma is not yet well-established as SE or SS type including correlation with angiogenesis. We classified canine SE and SS, and then compared them to tumor associated vessels., Methods: Twenty-three cases of canine seminomas (2 intratubular, 9 diffuse, and 12 intratubular/diffuse seminomas showing both intratubular and diffuse patterns) were classified as SE or SS by immunohistochemistry (IHC) using monoclonal antibody against PLAP and by PAS stain. The histopathological data were then compared to see if there was a correlation with SE or SS. Angiogenesis of seminomas were evaluated by immunohistochemical assay using polyclonal antibody against Von Willebrand factor (vWF) and by calculating the means of MVD, vessels area and perimeters using computerized image analysis. Statistical Package for Social Sciences (SPSS) program was used for various statistical analyses., Results: The numbers of PLAP+/PAS+ canine SEs were 8/23 (34.8%) and PLAP-/PAS- SSs were 15/23 (61.2%). All SE cases (8/8, 100%) were intratubular/diffuse types. SS types included 2 intratubular (2/15, 13.3%), 9 diffuse (9/15, 60%), and 4 intratubular/diffuse (4/15, 26.7%) types. MVD and vascular parameters in SEs were significantly higher than in SSs, showing the highest value in the intratubular/diffuse type. Seminomas observed with neoplastic cells invasion of vessels presented higher perimeter and area values than seminomas without conformed neoplastic cells invasion., Conclusion: In this study, we demonstrated a positive relationship between canine SE and tumor angiogenesis. Furthermore, we also showed that a tumor cells invasion of vessels were a correlated vascular parameter. Although metastasis of canine seminomas has rarely been reported, our results support that canine SE could have high metastatic potential similar to the human counterpart. Further studies are required to clarify the relationship between canine SE and clinical data with metastatic factors.

Background: The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas., Methods: The expression of the above genes was evaluated by means of quantitative RT-PCR, western blot, zymographic analysis and immunohistochemistry., Results: Quantitative RT-PCR analysis of 14 seminomas demonstrated that uPA and uPAR mRNAs were, with respect to control tissues, increased in tumor tissues by 3.80 +/- 0.74 (p < 0.01) and 6.25 +/- 1.18 (p < 0.01) fold, respectively. On the other hand, PAI-1 mRNA level was unchanged (1.02 +/- 0.24 fold), while that of PAI-2 was significantly reduced to 0.34 +/- 0.18 (p < 0.01) fold. Western blot experiments performed with protein extracts of three seminomas and normal tissues from the same patients showed that uPA protein levels were low or undetectable in normal tissues and induced in tumor tissues. On the same samples, zymographic analysis demonstrated increased uPA activity in tumor tissue extracts. Western blot experiments showed that also the uPAR protein was increased in tumor tissues by 1.83 +/- 0.15 fold (p < 0.01). The increased expression of uPA and uPAR was further confirmed by immunohistochemical staining performed in 10 seminomas and autologous uninvolved peritumoral tissues. Finally, variation in the mRNA level of PAI-1 significantly correlated with tumor size., Conclusions: We demonstrated the increased expression of uPA and uPAR in human seminomas with respect to normal testis tissues, which may be relevant in testicular cancer progression.

Necrotic testicular tumors are relatively frequent and can present a significant diagnostic challenge. Because of differing treatments for seminomas versus nonseminomas, accurate diagnosis is critical. Eleven totally (n=9) or almost totally (n=2) necrotic testicular tumors were retrieved from our consult files. The submitting pathologists favored benign processes in 4 cases, Leydig cell tumor in 1, and lymphoma in 1. The cases were evaluated for histologic features and, when material was available, by immunostaining with 7 antibodies: keratin (AE1/AE3), OCT4, placental alkaline phosphatase, alpha-fetoprotein (AFP), CD117, CD30, and S100. Only distinct reactivity in a cellular distribution in the necrotic zone was considered positive; nuclear reactivity alone was scored for OCT4 and membrane reactivity for CD117 and CD30. Mean patient age was 35 years (range 16-63). Mean tumor size was 19 mm (range 7-53). All patients presented with unilateral testicular masses (6 right, 5 left); 2 also had acute pain. The combination of histologic features, immunostains and, in 1 case, serum AFP permitted classification of 8 tumors (4 seminomas, 3 embryonal carcinomas, 1 yolk sac tumor). Three were not classifiable. The necrotic seminomas lacked associated coarse intratubular calcifications and were positive for OCT4 (4/4) and CD117 (3/3) but negative for keratin (0/4) and CD30 (0/4). The necrotic embryonal carcinomas had associated coarse intratubular calcifications and were positive for keratin (2/3), OCT4 (2/2), and CD30 (3/3). OCT4 stained 1 unclassifiable tumor, which lacked other specific markers. We did not find placental alkaline phosphatase, AFP, and S100 stains useful, although S100 did highlight tumor "ghost" cells in 1 case. Other features in most cases included intratubular germ cell neoplasia (6/11), tubular atrophy/hyalinization (10/11), tumor "ghost" cells (10/11), scar (9/11), and inflammation (10/11). Of the 5 patients with available follow-up, 3 were free of disease at 1, 5, and 8 years after orchiectomy (2 necrotic seminomas and 1 germ cell tumor, unclassified). One patient with yolk sac tumor (age 63 y) developed widespread metastases after 15 months and died of disease. The final case was initially misinterpreted as "testicular infarction, no malignancy" and 16 months later the patient developed a large retroperitoneal seminoma. Most totally necrotic testicular tumors can be placed into clinically important groups by assessment for coarse intratubular calcifications and staining reactions for keratin, OCT4, CD117, and CD30.

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1(-/-) mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.

Despite its alarming appearance, spermatocytic seminoma virtually never metastasizes. We hypothesized that this paradox may at least be partially related to increased apoptosis compared to metastasizing germ cell tumors since high expression of proapoptotic factors correlates with indolent behavior in other tumor systems, notably CD30-positive cutaneous lymphoma, another neoplasm where phenotype and behavior do not match. We therefore compared apoptosis and apoptotic regulators in 17 spermatocytic seminomas (2 with sarcoma) and 18 usual seminomas by light microscopy and using immunostains for caspase-3, p53, bcl-2, bcl-xL, FADD, FAS and survivin. We found significantly greater numbers of apoptotic cells and activated caspase-3-positive cells in spermatocytic seminoma compared to usual seminoma (P<0.01). There was over a 10-fold range in apoptotic cells in usual seminoma but only a 4-fold variation in spermatocytic seminoma. Spermatocytic seminoma had decreased p53 expression compared to usual seminoma, with marked variation in bcl-2 expression and increased FADD. The two sarcomas in spermatocytic seminoma, however, showed decreased apoptosis and caspase-3 reactivity, with upregulation of p53 and bcl-2 and decreased FADD expression. We conclude that apoptosis, caspase-3 and FADD expression are increased in spermatocytic seminoma compared to usual seminoma. Apoptotic parameters are decreased in sarcomatous transformation of spermatocytic seminoma. The increased apoptosis of spermatocytic seminoma, possibly mediated by FAS independent activation of the death receptor pathway, may provide some insight into its excellent prognosis. The variation in apoptosis of usual seminomas merits investigation as a prognostic parameter.

The classification of intratubular germ cell neoplasia of the testis includes an unclassified type (IGCNU), in addition to various other intratubular lesions that show specific forms of differentiation, such as intratubular seminoma and intratubular embryonal carcinoma. Although IGCNU is recognized as a precursor lesion for testicular germ cell tumors, the relationship between differentiated types of intratubular germ cell neoplasia and invasive germ cell tumors of the testis is not well established. The aim of the present study was to examine the association between invasive testicular germ cell tumors and intratubular neoplastic lesions, with particular emphasis on differentiated types of intratubular germ cell neoplasia. The seminiferous tubules adjacent to 42 testicular germ cell tumors were evaluated for the presence of various forms of intratubular germ cell neoplasia. IGCNU was observed in 37 (88%) of 42 cases, whereas intratubular seminoma and intratubular embryonal carcinoma were seen in 19% and 7% of the cases, respectively. Intratubular seminoma was associated primarily with seminomas or mixed germ cell tumors with a seminomatous component, but was also present in a case of a nonseminomatous germ cell tumor. Intratubular embryonal carcinoma was associated exclusively with nonseminomatous germ cell tumors. All cases of intratubular embryonal carcinoma were identified morphologically and exhibited histologic features corresponding to traditional definitions of this lesion. No examples of intratubular embryonal carcinoma as defined by CD30 expression alone in the absence of an intratubular proliferation were observed. The presence of intratubular seminoma in a nonseminomatous germ cell tumor suggests that it is a true preinvasive lesion rather than a manifestation of intratubular spread of an established invasive seminoma. The low incidence of intratubular embryonal carcinoma supports the theory that most nonseminomatous germ cell tumors evolve initially as seminomas, rather than directly from a differentiated intratubular neoplastic lesion.

IGF2 and H19 are imprinted genes in normal human tissue, but many studies have observed a loss of imprinting (LOI) of these genes in tumors as an epigenetic alteration of the DNA, that leads to a biallelic expression predisposing cells to carcinogenesis and tumor growth. The aim of this study was to test the reliability of LightCycler-assisted Real-time PCR in detecting LOI of IGF2 and H19 in 39 patients with testicular germ cell tumors by comparing these results with the analysis generated by the golden standard restriction fragment length polymorphism (RFLP). With LightCycler-assisted Real-time PCR for IGF2 44% and for H19 49% of the patients were found to be heterozygous. This was consistent with the results obtained by RFLP, but surprisingly RFLP failed in more than 7% of the patients. In detecting LOI (for IGF2 in 41% and for H19 in 68% of the informative patients) the approach by RFLP was superior, since the results derived from LightCycler-assisted Real-time PCR showed reliable results in 76 and 10% of the samples concerning IGF2 and H19, respectively. Again, no discrepancy between the results obtained by the two methods occurred. In sum, LightCycler-assisted Real-time PCR is a sufficiently working approach for the rapid and reliable detection of heterozygosity of IGF2 or H19 gene and identification of LOI of IGF2 and thus may be helpful in conducting large epidemiological studies. However, for the identification of LOI of the H19 gene in this cohort it possesses only restrictive use.

Intratubular seminoma (ITS) has been defined as the complete filling of the seminiferous tubules with seminoma cells with no Sertoli cells present. This contrasts with intratubular germ cell neoplasia, unclassified (IGCNU), where the malignant germ cells are interspersed by Sertoli cells. We aimed to determine the relationship between these 2 entities and the association between ITS and invasive classic seminomas. We therefore examined the morphology and immunochemistry of ITS and IGCNU adjacent to germ cell tumors to differentiate the patterns, frequency, and distribution of these lesions. We found that ITS was seen in equal frequency adjacent to seminomas as it was to nonseminomas. The presence of ITS in non-seminomatous germ cell tumors suggests that it is a true in situ lesion rather than representative of intratubular spread of an existing seminoma. However, because it is not specifically associated with seminoma, we suggest that it is not useful to discriminate this lesion from IGCNU and that it merely represents an advanced form of IGCNU on the way to invasive malignancy.

We report 28 testicular seminomas with cystic spaces of variable nature, sometimes accompanied by solid and hollow tubular patterns (12 cases). The spaces often suggested reticular or microcystic patterns of yolk sac tumor, and the solid and hollow tubular patterns often added to the diagnostic confusion. The tumors occurred in men 21 to 55 years old and on gross examination had the typical appearance of seminoma. On microscopic examination, the spaces ranged from small, closely packed and relatively regular to dilated, more dispersed and somewhat irregular. The hollow tubules appeared to result from central discohesion within nests of tumor. The spaces, particularly when large, often contained occasional tumor cells or inflammatory cells within pale edema fluid. The cytologic appearance of the cells lining the spaces, and in the surrounding tumor, retained the typical features of seminoma cells. Thirteen tumors (46%) either lacked (8 cases) or had very scant (5 cases) lymphocytes in the cystic and tubular areas, and hyaline globules were absent. Thirteen of 13 tumors were immunopositive for OCT-3/4 in the nontypical and typical areas; 9 of 10 were placental alkaline phosphatase positive, and 7 of 10 were c-Kit (CD117) positive. The same 13 cases were negative with cytokeratin (AE1/AE3) and alpha-fetoprotein stains. Distinction from yolk sac tumor is aided by the observation that the spaces of yolk sac tumor are often more irregular in their individual shapes and frequently form anastomosing channels. Additionally, the spaces of yolk sac tumor randomly merge with various other yolk sac tumor patterns. The cells lining spaces in yolk sac tumor are often flattened with compressed nuclei and lack the typical prominent nucleoli of seminoma cells. Paucity of lymphocytes and intracystic edema, however, are not differentially helpful, although basophilic fluid favors yolk sac tumor. A panel of immunostains (AE1/AE3, OCT-3/4, and alpha-fetoprotein) is helpful in the differential with yolk sac tumor in especially problematic cases. The edema and paucity of lymphocytes may suggest spermatocytic seminoma, but the varied cell types of that neoplasm are absent.

The drug-efflux pumps P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) are present in the blood-testis barrier (BTB) and may hamper the delivery of cytotoxic drugs to the testis. The precise localisation of P-gp and MRP1 in testicular tissue and the presence of the efflux pumps MRP2 and breast cancer resistance protein (BCRP) in the BTB are unknown. We therefore studied the localisation of these pumps in the BTB in normal testis (n = 12), in non-seminoma (n = 10) seminoma (n = 10), and testicular lymphoma (n = 9). Slides were scored semi-quantitatively for P-gp, MRP1, MRP2 and BCRP and blood vessels with factor VIII antibody. In normal testis, P-gp and BCRP were strongly expressed by myoid cells and luminal capillary endothelial wall and P-gp also by Leydig cells. MRP1 was observed at the basal side of Sertoli cells and on Leydig cells. MRP2 was only weakly expressed by myoid cells. Seminomas and non-seminomas expressed P-gp and/or BCRP and/or MRP1, lymphomas strongly expressed P-gp, weakly expressed BCRP and did not or showed weak expression of MRP1. There was very little staining for MRP2 in the tumours. Newly formed vessels in all tumours only expressed P-gp and BCRP. P-gp, BCRP and MRP1 are present in different cell layers of the normal testis, suggesting the optimal protection of spermatogenesis. In germ cell tumours, this expression pattern may explain the chemoresistance observed to P-gp, BCRP and MRP1 substrates. In germ cell tumours and testicular lymphomas, P-gp and BCRP expression by tumour cells and by newly formed vessels may also contribute to chemoresistance. These findings underscore the importance of removing the affected testis in cases of primary germ cell tumours and testicular lymphomas, irrespective of whether the patient has already undergone chemotherapy.

OCT4 (POU5F1) is a transcription factor expressed in embryonic stem and germ cells and is involved in the regulation and maintenance of pluripotency. It has been detected in primary testicular germ cell tumors with pluripotent potential, seminoma, and embryonal carcinoma. We undertook immunohistochemical staining of OCT4 in a wide variety of primary testicular neoplasms (germ cell tumors and other tumors) to assess the specificity and usefulness of this marker as a diagnostic tool. We examined histologic sections from 91 primary testicular neoplasms, including 64 cases of mixed germ cell tumors containing embryonal carcinoma (54), seminoma (51), yolk sac tumor (38), mature teratoma (31), immature teratoma (20), and choriocarcinoma (15). In addition, we examined sections from spermatocytic seminomas (5), Leydig cell tumors (8), Sertoli cell tumors (6), unclassified sex-cord stromal tumors (4), adenomatoid tumors (2), testicular tumor of adrenogenital syndrome (1), and granulosa cell tumor (1). Each tumor was examined with hematoxylin and eosin staining and with antibodies to OCT4. In all cases of mixed germ cell tumor with components of embryonal carcinoma (54) and seminoma (51), there was greater than 90% nuclear staining of the embryonal carcinoma and seminoma tumor cells with little to no background staining. In all but 1 of these cases (embryonal carcinoma), there was strong (3+) staining intensity. The other germ cell tumor components (yolk sac tumor, mature teratoma, immature teratoma, and choriocarcinoma) showed no staining. Syncytiotrophoblast cells, which were present in 15 of the cases, were also completely negative, as were all 5 of the spermatocytic seminomas. The 22 cases of non-germ cell tumors were all immunohistochemically negative for OCT4. Fifteen of the 54 germ cell tumors containing embryonal carcinoma were also examined with antibodies to CD30. These embryonal carcinoma components were all positive for CD30 with staining of greater than 90% of the tumor cells but with variable staining intensity. We conclude that immunostaining with antibodies to OCT4 is a useful diagnostic tool in the identification of primary testicular embryonal carcinomas and "usual," but not spermatocytic, seminomas. OCT4 immunostaining has comparable sensitivity but greater consistency compared with CD30 in the diagnosis of embryonal carcinoma.

We report the case of a 17-yr-old male with ambiguous genitalia, 45,X/46,XY mosaic karyotype, and Y chromosome microdeletions. The patient underwent a testicular biopsy at the age of 6 with normal findings. A second biopsy at the age of 17 established the diagnosis of intratubular germ cell neoplasia (ITGCN), which was treated with bilateral orchidectomy. This case report deals with three important issues regarding ITGCN: First, although a prepubertal biopsy can be performed in order to provide evidence for future fertility, it is very unreliable for making a diagnosis of ITGCN. Second, because ITGCN tends to be a generalized procedure that affects both testes in a uniform pattern, a small number of biopsies, even a single one, could be adequate for diagnostic purposes in the majority of cases. Third, although the population that requires screening for ITGCN remains controversial, the early postpubertal period could be the optimum time for a testicular biopsy.

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.

The distinction of Sertoli cell tumors from seminoma is critical to ensure proper treatment. Although usually straightforward, we highlight herein 13 malignant Sertoli cell tumors of the testis with light microscopic features that mimicked seminoma. All of the cases were received in consultation, 10 with submitting diagnoses of seminoma, usually of classic type, but three cases of spermatocytic type. Patients ranged from 15 to 80 years of age (median 37 years); all presented with testicular masses. The tumors were typically firm, white to yellow-tan, and often had foci of hemorrhage. The dominant microscopic pattern was nested or sheet-like, with some tumors having secondary patterns of trabeculae-solid tubules, hollow tubules, and pseudofollicles. Tumor cells were polygonal with conspicuous clear cytoplasm in 12 cases; the cytoplasm was focally eosinophilic in 10 cases, but this was never conspicuous. Nine tumors had cytoplasmic vacuoles, and three of four that were investigated stained for intracytoplasmic glycogen. Nuclei were small (5) to medium-sized (8), round-to-oval (13), and vesicular with irregular contours (11). Nucleoli were present in 11 tumors (six small; five large). Stromal fibrosis (12) and lymphoid infiltrates (10) were conspicuous, and tumor necrosis (11) and vascular invasion (8) also were seen. Mitotic figures ranged from <1 to 21/10 high power fields (HPF) (median 1/10 HPF). Staining for inhibin-alpha, epithelial membrane antigen, and cytokeratin (AE1/AE3) was positive in four of four, six of six, and three of six cases, respectively; placental alkaline phosphatase was negative in all five tumors investigated. The nested growth pattern, prominence of clear cells, lymphoid infiltrate, inconspicuous tubular differentiation, cytoplasmic glycogen, and prominent nucleoli caused these tumors to be mistaken for seminomas. The smaller, less pleomorphic nuclei of Sertoli cell tumors, their lower mitotic rate, and the absence of intratubular germ cell neoplasia are helpful differential features. Immunohistochemistry is a useful adjunct in confirming the diagnosis of Sertoli cell tumor, but only if the overlapping features are appreciated by conventional microscopy and the diagnosis of Sertoli cell tumor included in the differential.

In this article we report on our diagnostic experience of fine-needle aspiration biopsies (FNAB) performed on 17 patients with testicular lesions in the period from 1994-1998. The cytological diagnosis was consistent with seminoma in 7 cases, sex cord-stromal tumors in 3 cases (2 Sertoli cell tumors, 1 Leydig cell tumor), embryonal carcinoma in 3 cases, and yolk-sac tumor in 1 case; the other 3 patients were suffering from flogistic pathology. The cytological diagnosis was confirmed in all cases after surgery. According to our experience, ultrasound FNAB of testicular lesions proved to be a very reliable technique in predicting malignancy with high sensitivity and specificity. None of the patients developed local recurrences or inguinal lymph-node metastasis due to FNAB. Therefore, tumor stage classification (TNM) was not modified in any patient., (Copyright 2000 Wiley-Liss, Inc.)

The gonadoblastoma locus on the Y chromosome (GBY) predisposes the dysgenetic gonads of XY females to develop in situ tumors. It has been mapped to a critical interval on the short arm and adjacent centromeric region on the Y chromosome. Currently there are five functional genes identified on the GBY critical region, thereby providing likely candidates for this cancer predisposition locus. To evaluate the candidacy of one of these five genes, testis-specific protein Y-encoded (TSPY), as the gene for GBY, expression patterns of TSPY in four gonadoblastoma from three patients were analyzed by immunohistochemistry using a TSPY specific antibody. Results from this study showed that TSPY was preferentially expressed in tumor germ cells of all gonadoblastoma specimens. Additional study on two cases of testicular seminoma demonstrated that TSPY was also abundantly expressed in all stages of these germ cell tumors. The present observations suggest that TSPY may either be involved in the oncogenesis of or be a useful marker for both types of germ cell tumors., (Copyright 2001 S. Karger AG, Basel)

Concentrations of two small stress proteins, alphaB crystallin and the 27-kDa heat shock protein (HSP27) were quantitated in tissues of the human normal genitourinary system and their tumors. Levels of HSP27 in renal cell carcinomas (mean +/- SE: 1450+/-262 ng/mg protein, n = 15) were significantly higher than in normal kidney (the cortex: 540+/-99 ng/ mg protein, n = 13; the medulla: 600+/-106 ng/mg protein, n = 13) while those of alphaB crystallin tended to be increased without statistical significance. These findings were similar to those previously reported for renal cell tumors chemically induced in rats. Concentrations of alphaB crystallin in prostatic carcinoma tissues (410+/-129 ng/mg protein, n = 10) were also significantly higher than in benign prostatic hyperplasia (54+/-12 ng/mg protein, n = 14), whereas alphaB crystallin levels in testicular tumors including seminomas (2.1+/-0.8 ng/mg protein, n = 11) and non-seminomas (5.2+/-2.3 ng/mg protein, n = 9) were significantly lower than in normal testicular tissues (29.7+/-6.2 ng/mg protein, n = 5). Both alphaB crystallin and HSP27 could be immunohistochemically localized in the normal kidney and renal cell carcinoma tissues.

Testicular anaplastic seminoma, which has a high mitotic activity, is regarded as more malignant than typical seminoma, although its prognosis is still unclear. To determine whether seminoma with relatively greater malignancy potential can be identified based on the cell proliferative activity, the mitotic rate (MR; mitotic count per high-power field), mitotic index (MI; mitotic count per 1000 cells), Ki-67 labeling index (Ki-67 LI; the percentage of positive cells) and proliferating cell nuclear antigen LI (PCNA LI; the percentage of positive cells) were histologically examined in 44 patients. The MI, Ki-67 LI and PCNA LI in patients with metastatic disease were significantly higher than those in patients without metastatic disease, and the MI in patients with fatal disease was significantly higher than those in patients cured of the disease. However, these distributions of the MI, Ki-67 LI and PCNA LI values overlapped for both pairs of groups. There were no significant differences in the MR. These results suggest that the cell proliferative activity makes a small contribution to the malignancy potential in testicular seminoma, with the activity being not necessarily indicative of metastasis and prognosis.

Primary mediastinal seminomas (MS) are rare tumors. Histologically, they are similar to their counterpart in the gonads. The survival rate has varied from 50% to 85% in different series. However, large series of these tumors primarily in the mediastinum are lacking. At the molecular level, a few reports document K-ras mutations in up to 40% of testicular seminomas (TS), localized predominantly to codon 12. Reports on TS p53 immunohistochemistry (IHC) range from negative to overexpression approaching 90% of cases, and by sequence analysis one small series showed a 23% mutation rate. To date, no analyses have been performed for either K-ras mutations or p53 immunohistochemical expression in primary MS. The authors studied 13 cases each of primary MS and TS from archival formalin-fixed, paraffin-embedded sections in which adequate tumor sampling and clinical history, including serological studies, and histological, histochemical, and IHC staining, were performed to confirm the diagnosis. p53 immunoperoxidase staining using citrate buffer/microwave antigen retrieval was performed. Topographic genotyping was performed on 5-microns-thick tissue sections up to 17 years old, in which the neoplastic cell population was sampled. Additionally, multiple sites within a given cases were sampled to determine clonality of the tumor cell population. Polymerase chain reaction and subsequent sequence analysis of the K-ras-2 exon-1 gene was used for mutation analysis. Focal weak staining with p53 IHC was observed in 4 of 13 (31%) MS and 10 of 13 (77%) TS cases, with all remaining cases being negative (P < .05). Only one MS case (8%) showed K-ras mutation (codon 13 GGC > GAC; glycine > aspartate), which is in contrast to 2 of the TS cases (15%), showing codon 12 mutations. All the remaining cases were wild type. Therefore, primary mediastinal seminomas appear to be different in their K-ras sequence and p53 immunostain profile from TS. Codon mutation type may be useful in determining primary versus metastatic origin of a mediastinal seminoma.

Background: Altered glycosylation is a common phenotype expressed in essentially all types of human cancer and has been found to be correlated closely with the invasive and metastatic properties of a given tumor. Because there was no prognostic information concerning aberrant glycosylation of seminoma, the authors studied this topic., Methods: Glycosphingolipid (GSL) composition of orchiectomy samples of seminoma were analyzed systematically. GSL patterns from seminoma samples of the following three groups were compared after a 44-month postoperative period: Stage I disease with no evidence of metastasis during the 44-month postoperative period, Stage I with metastatic relapse during this period, and Stage II with retroperitoneal lymph node metastasis. Unknown GSLs detected were analyzed chemically by 1H-nuclear magnetic resonance spectroscopy and mass spectrometry., Results: All nonmetastatic seminomas (n = 12) contained a GSL band that was identified as galactosylgloboside (Gb5; Gal beta 1-->3GalNAc beta 1-->3Gal alpha 1-->4 Gal beta 1-->4Glc beta 1-->1Cer). All metastatic seminomas (n = 5) lacked this GSL, although the sample sizes were admittedly small., Conclusion: Only the presence or absence of galactosylgloboside (Gb5), but of no other GSL or gangliosides, clearly correlated with metastatic potential in patients with seminoma. This observation is useful in the estimation of prognosis of patients with seminoma, especially those with Stage I disease.

We present a strategy for the evaluation of numerical copy number changes of DNA segments within a solid tumor genome that allows the correlation of microscopic phenotype with genotype in formalin-fixed, paraffin-embedded tumor material. Cells from a human testicular germ cell tumor and adjacent tissue areas with normal seminiferous tubules were selected separately from microscopically analyzed histological tissue sections, and DNA was extracted from the selected areas. After universal DNA amplification, the amplification products were subjected to comparative genomic hybridization. The results confirmed balanced chromosome copy numbers for the normal tissue area, although the analysis of the tumor tissue area revealed numerous gains and losses of chromosome segments. The comparative genomic hybridization results were used to select DNA probes for interphase cytogenetics on serial sections. We conclude that this technique allows the screening of selected tissue areas for numerical DNA alterations, thus enabling a direct phenotype-genotype comparison.

The expression of laminin was studied to determine the distribution pattern of basement membranes (BMs) in normal testes and in a series of 40 canine testicular tumours (seminomas, Leydig and Sertoli cell tumours). BM was always present around seminiferous tubules and blood vessels in normal testes and in seminomas and Sertoli cell tumours of the intratubular type without invasion. BM changes (fragmentation or loss, or both) were usually found in invasive neoplasms which retained their tubular structure; disruption or absence was observed in tumours, with a diffuse pattern. The BM was never expressed in Leydig cell tumours, except around vessels, irrespective of their histological growth pattern (cystic-vascular, pseudoadenomatous, diffuse). An attempt was made to relate the degree of BM modification to proliferative monoclonal antibodies and mitotic index. In parallel with the progressive loss of BM an increase in proliferative activity occurred, indicating that BM changes are additional useful prognostic indicators in testicular tumours of the dog.

In the present study, we examined the distribution of integrin subunits and extracellular matrix proteins in normal testis, intratubular germ cell neoplasia (ITGCN), and primary and metastatic seminomas. Compared to normal testis in ITGCN, Sertoli cells showed increased expression of alpha 3, alpha 6, and beta 1 integrin subunits. Malignant intratubular germ cells stained for alpha 3, alpha 6, and beta 1 integrin subunits. Progression of ITGCN to invasive seminoma was associated with loss of alpha 3 integrin subunit expression by tumor cells. Consequent to this loss, it can be speculated that the strong expression on ITGCN may be related to the noninvasive character of the lesion as is also known from other noninvasive tumors. All tumors showed a strong expression of alpha 6 and beta 1 integrin subunits. The alpha 5 integrin subunit was weakly expressed in primary seminomas in all stages. No differences were observed in integrin expression between primary and metastatic tumors. The distribution of extracellular matrix proteins was heterogeneous and revealed clear architectural differences between seminomas that may reflect different stages of tumor stroma formation. To our knowledge, the results presented in this study provide the first information on the possible role of tumor-extracellular matrix interactions in the biological behavior of ITGCN and testicular seminomas.

Eighteen hundred and eighty-four cases of human solid tumours and 833 samples of normal human tissues, formalin-fixed and paraffin-embedded, were examined immunohistochemically for expression of c-kit oncogene product using polyclonal antibody against synthesized c-kit peptide. Seminoma/dysgerminoma and small cell lung carcinoma (SCLC) show preferential c-kit expression at 92% and 36% frequency, respectively, whereas only sporadic cases of cervical carcinoma and non-SCLC lung carcinoma show c-kit positivity. A normal tissue counterpart positive for c-kit product is detected in the testis (spermatocyte) and ovary (oocyte) but not in the lung or the cervix. In contrast, normal epithelial cells of the breast, skin basal cells and tissue mast cells harbour c-kit product, but transformed cells of the former two are largely deficient in the c-kit protein. One hundred and thirty-nine neuroendocrine tumours and 39 non-pulmonary small cell carcinomas were all negative, except for two cases of neuroblastoma. This indicates a distinct character for SCLC in c-kit expression. The c-kit product may be a useful marker in diagnostic pathology of seminoma/dysgerminoma and SCLC among human solid tumours, and in distinction of SCLC from non-pulmonary small cell carcinoma.

In order to study the possible biological differences between anaplastic and typical seminoma, the following factors were studied in 11 cases of anaplastic seminoma and 15 cases of typical seminoma: mitotic activity, proliferating cell nuclear antigen (PCNA) expression, immunohistochemical analyses for cytokeratin, vimentin, placental alkaline phosphatase (PLAP), beta-human chorionic gonadotropin (beta-hCG), alpha-fetoprotein (AFP) and c-myc oncoprotein. Anaplastic seminoma was classified according to Mostofi's criteria, which is primarily based on the mitotic activity of the tumor. Mitotic activity was evaluated by both mitotic count and rate. Statistically significant correlations were observed between mitotic count and mitotic rate (R = 0.891), and between the mitotic count and PCNA labeling index (R = 0.792), in both typical and anaplastic seminomas. Immunostaining patterns for cytokeratin, vimentin, PLAP, beta-hCG, AFP and c-myc oncoprotein were not significantly different between typical and anaplastic seminoma. The present data indicated that no apparent clinicopathologic and immunohistochemical parameters discerning anaplastic seminoma from typical seminoma were present, when identifying anaplastic seminoma on the basis of high mitotic count. Anaplastic seminoma may therefore simply represent seminoma with high proliferative activity.

Characterization of the nuclear DNA content (DNA index and DNA histogram type) was carried out in 9 normal testicular tissues and 21 seminomas. Proliferative activity was further determined in the seminomas. Nuclear DNA content was assessed by means of a cell image processor computing the integrated optical density on Feulgen-stained nuclei from formalin-fixed paraffin-embedded materials. The results indicated that the so-called normal testicular tissues exhibited DNA histograms where three cell nuclei populations emerged in varying proportions. These three cell nuclei populations corresponded to haploid, diploid and tetraploid cell nuclei respectively. In contrast, most of the seminomas exhibited monomorphic DNA histogram patterns with a predominance of GO-G1 cell nuclei in the range of the 3c-4c nuclear DNA content. Further studies will be necessary in order to determine whether the DNA histogram has a predictive prognostic value or could be considered as a grading index.

A case of a spontaneous intratubular spermatic seminoma is described in a 98-wk-old Fischer-344 (F-344) rat. The differential diagnosis of spermatic seminoma from the other morphological forms of the tumor was based on the recognition of 3 cell types. The tumor cells were positive for S-100 antigen and cytokeratin. To the best of our knowledge, this is the second case of a seminoma reported in an F-344 rat and the first dealing with electron microscopic and immunohistochemical features of this tumor.