We found that benign lesions represent the majority of primary testis tumors (74%), with the most common histological type being teratoma (48%). The reported high prevalence rates of prepubertal yolk sac tumors probably results from a reporting bias, since benign tumors are less likely to be submitted to tumor registries. Therefore, the primary operative approach to the majority of testis tumors in boys 12 years and younger should entail testis sparing surgery. Orchiectomy should be reserved for histologically confirmed malignancy based on increased preoperative alpha-fetoprotein and/or frozen section analysis of the tumor.

A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.

Background: Previous studies suggest that two fundamental, probably androgen-dependent, steps in maturation of germ cells normally occur in the prepubertal testis: the disappearance of gonocytes (the fetal stem cell pool) and the appearance of adult dark spermatogonia (the adult stem cell pool) at 2–3 months of age and the appearance of primary spermatocytes (the onset of meiosis) at 4–5 years. Previous studies of small series of cryptorchid boys suggest that both steps are defective in undescended testes and to a lesser degree in descended testes contralateral to unilaterally undescended testes. The purpose of this study is to confirm the previous findings of defective germ cell maturation in a large series of boys with unilateral undescended testes. Patients: Seven hundred and sixty-seven boys with unilateral cryptorchidism who had orchidopexy and bilateral testicular biopsies between birth and 9 years of age were studied. Materials and Methods: Total and differential germ cell counts were performed on semithin histologic sections of the biopsies. The results from the undescended and contralateral descended testes were compared using the Wilcoxon signed-rank test and the Wilcoxon-Whitney-Mann U test. Results: Gonocytes failed to disappear and adult dark spermatogonia failed to appear in undescended testes under 1 year of age indicating a defect in the first step in maturation at 2–3 months resulting in failure to establish an adequate adult stem cell pool. Primary spermatocytes failed to appear in undescended testes and appeared in only 19% of contralateral descended testes at 4–5 years of age indicating a defect in the onset of meiosis. Conclusion: Unilaterally undescended testes fail to establish an adequate adult stem cell pool which normally occurs at 2–3 months of age and fail to establish adequate meiosis which normally occurs at 4–5 years of age. Similar but less severe changes are seen in the contralateral descended testes. Defects in the two pubertal steps in germ cell maturation are associated with reduced total germ cell counts.

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Unlike previously published series based on tumor registries, benign teratoma was the most common pediatric testicular tumor treated at our institution. Our single institution experience with testis preservation and long-term followup confirms the role and safety of this technique. Testis sparing surgery remains our technique of choice for testicular teratoma.

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The WT1 gene encodes a transcription factor implicated in normal and neoplastic development. The purpose of this study was to evaluate the diagnostic utility of a commercial WT1 antibody on a variety of pediatric small round blue cell tumors (SRBCT). A mouse monoclonal antibody (clone: 6F-H2, DAKO) raised against the N-terminal amino acids 1-181 of the human WT1 protein was tested. Microscopic sections from 66 specimens were stained using an antigen retrieval protocol with trypsin. The tumors included peripheral neuroectodermal tumors (PNET/Ewing's), neuroblastomas, desmoplastic small round cell tumors (DSRCT), lymphomas, Wilms' tumors, and rhabdomyosarcomas (RMS). One RMS case was investigated by Western blot analysis and RT-PCR to confirm the antibody specificity. A strong cytoplasmic staining was demonstrated in all RMS (11/11). The Western blot analysis confirmed the WT1 protein in the tissue, and the RT-PCR confirmed the presence of WT1 mRNA in the peripheral blood and tissue of one RMS patient. The Wilms' tumors had a variable nuclear and/or cytoplasmic positivity in most (17/24) cases. All PNET/Ewing's were negative. The nuclei of two lymphoblastic lymphomas stained strongly. A weak nuclear or cytoplasmic staining was reported in a few DSRCT (3/5), lymphomas (2/10), and neuroblastomas (2/8). This is a useful antibody in the differentiation of RMS from other SRBCTs. A strong cytoplasmic staining favors an RMS, and a strong nuclear staining is suggestive of a Wilms' tumor. A role for WT1 in the pathogenesis of rhabdomyosarcomas is raised. The limited sampling precludes any conclusions regarding the value of tissue or peripheral blood analysis for WT1 mRNA in patients with rhabdomyosarcoma. The WT1 gene (1) encodes a protein with four zinc fingers of the Kruppel-type in the C-terminal region that recognizes a guanidine-cytidine (GC)-rich "EGR1" consensus sequence (2) required in tissue differentiation and proliferation (2-4). The N-terminal half contains a large proline-glutaminerich domain important for inhibition of transcriptional activation (5, 6). There are at least eight protein isoforms ranging between 52 and 62 kDa in mammals produced by a combination of alternative splicing and RNA editing (4, 7). The WT1 proteins are normally expressed in the nuclei of glomerular podocytes and mesothelial cells. It has also been demonstrated in stem cells bearing the CD34ϩ phenotype (8). The role of WT1 in normal human development also extends to a diversity of mammalian mesodermal tissues (9), including the body-wall musculature in a 13.5-days postconception (dpc) mouse embryo (43-49 dpc human). Embryologic studies of wt1-null mice reveal a failure to develop kidney and gonads (10). Mutations and splicing disruptions of WT1 have been described in , WAGR (15), and Frasier (13,16) (17-22), mesothelial-derived neoplasms (23-27), breast cancer (28, 29), genitourinary tumors (30, 31), and small round blue cell tumors (SRBCT;24,27,(32)(33)(34). Recent studies have evaluated the possible role of peripheral blood RNA ...

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