Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks§.

"Differences of Sexual Development (DSD)," individuals with rearranged Y chromosome breaks in their 46,XY cells are reported with male and female gender phenotypes and differences in germ cell tumour (GCT) risk. This raised the question of whether male or female gender and GCT risk depends on the site of the break and/or rearrangement of the individual´s Y chromosome. In this paper, we report molecular mapping of the breakpoint on the aberrant Y chromosome of 22 DSD individuals with a 45,X/46,XY karyotype reared with a different gender. Their Y chromosome breaks are found at different sites on the long and short Y arms. Our data indicate that gender rearing is, neither dependent on the site of Y breakage, nor on the amount of 45,X0 cells in the individuals' leukocytes. Most prominent are secondary rearrangements of the Y chromosome breaks forming di-centric Y-structures ("dic-Y"). Duplications of the short Y arm and the proximal part of the long Y arm are the results. A putative GCT risk has been analysed with immunohistochemical experiments on some dysgenetic gonadal tissue sections. With specific antibodies for OCT3/4 expression, we marked the pluripotent germ cell fraction being potential tumour precursor cells. With specific antibodies for DDX3Y, TSPY, and UTY we analyzed their putative Gonadoblastoma Y (GBY) tumour susceptibility function in the same specimen. We conclude GBY expression is only diagnostic for GCT development in the aberrant germ cells of these DSD individuals when strong OCT3/4 expression has marked their pluripotency.

Gonadoblastoma: origin and outcome.

Classical gonadoblastoma occurs almost entirely in the dysgenetic gonads of an individual who has a disorder of sex development; however, a small number of cases arise in individuals with a normal peripheral karyotype and no evidence of a disorder of sex development. Those gonadoblastomas that occur in an individual who has a Y chromosome or part thereof express testis specific protein Y-encoded 1 (TSPY1). If a gonad in those individuals contains germ cells with delayed maturation and also harbors the TSPY1 gene, the cells can undergo transformation to classical gonadoblastoma. The latter consists of rounded islands composed of germ cells, sex cord elements, and hyaline basement membrane material surrounded by a variably cellular stroma that sometimes contains steroid cells. Classical gonadoblastoma can be interpreted as a noninvasive or an in situ neoplasm that is the precursor of germinoma in some individuals and, indirectly, of other more aggressive germ cell neoplasms. The "dissecting" variant is derived from classical gonadoblastoma and is characterized by unusual growth patterns. Undifferentiated gonadal tissue is the precursor of gonadoblastoma; however, if all germ cells in an individual with undifferentiated gonadal tissue involute, the result is a secondary streak gonad. Undifferentiated gonadal tissue is a non-neoplastic condition resembling a streak gonad but additionally contains germ cells with delayed maturation that express octamer-binding transcription factor 4; however, other germ cells, show normal maturation and express TSPY1.

Usefulness of imprint cytology of gonadoblastoma with dysgerminoma in a patient with Turner syndrome and a Y chromosome: A case report and literature review.

Ovarian gonadoblastoma coexisting with a dysgerminoma is extremely rare in patients with Turner syndrome (TS) and a Y chromosome. The cytological findings, including imprint cytology, of these unusual ovarian tumors have rarely been reported. We report a rare patient with a gonadoblastoma with dysgerminoma, 3.0 × 2.0 cm in size; she was a 19-year-old woman with TS and a Y chromosome. She underwent laparoscopic bilateral gonadectomy, and the tumor was classified as stage IA (pT1aNxM0) according to the International Federation of Gynecology and Obstetrics classification system. Intraoperative imprint cytology revealed two types of neoplastic cells: small tumor cells surrounding light green-stained or eosinophilic hyaline globules with marked calcification, suspicious for gonadoblastoma; and large, round, atypical cells with abundant cytoplasm, macronucleoli, and marked lymphocytic infiltration (two-cell pattern), suspicious for dysgerminoma. The cytology results in our patient may represent the second reported results of imprint cytology describing a gonadoblastoma with dysgerminoma. They are the first reported results in a patient with TS and a Y chromosome.

"Dissecting Gonadoblastoma" of Scully: A Morphologic Variant That Often Mimics Germinoma.

Dr Robert E. Scully, who recognized and defined gonadoblastoma (GB), used the term "dissecting gonadoblastoma" (DGB) to describe variants with either an infiltrative type or diffuse pattern instead of the usual small nested arrangement. These patterns have not been emphasized in the literature. To investigate the features of DGB we examined 50 GBs microscopically and performed immunohistochemistry (IHC) in some. DGB was found in 38 (76%) GBs and was represented by 3 patterns. The most frequent was solid/expansile (n=26), consisting of large coalescent nests of germ cells, often (92%) interrupted by fibrovascular septa, with usually minor numbers of sex cord cells. Less frequent were small anastomosing nests (n=24) and cord-like arrangements (n=22) of germ cells irregularly distributed in a prominent stroma and with mostly inconspicuous sex cord cells. Most DGBs (24) showed >1 pattern and demonstrated the characteristic globular deposits of basement membrane, although these were often subtle. The germ cells in all patterns varied from spermatogonium-like to seminoma-like; OCT3/4 was positive only in the latter (7/7). The sex cord cells were small with dense, oval or angulated nuclei, inconspicuous nucleoli, and positivity for inhibin (9/9, strong), FOXL2 (9/9, strong), SF1 (8/9, strong), SOX9 (9/9, weak and focal), WT1 (5/7, variable), and calretinin (3/7, variable). Granulomas were present in 84% of germinoma foci, 13% of DGB foci, and 8% of classic GB foci. Twenty two of 38 DGBs had associated germinoma; 3 also had embryonal carcinoma, yolk sac tumor, and choriocarcinoma, respectively. Follow-up of 2 cases lacking an invasive tumor showed that both patients were disease free at 13 and 4.8 years after bilateral gonadectomy. We conclude that DGB is commonly seen with classic GB and displays identical IHC features, supporting it as a morphologic variant of GB. It appears likely that cord-like DGB is the earliest phase in a GB developmental continuum that may proceed successively into anastomosing, nested (classic GB), and solid/expansile patterns. DGB often mimics germinoma because of the large size of the nests, pseudoinfiltrative pattern of some cases, and inconspicuous sex cord cells. The presence of sex cord cells (identification aided by IHC for sex cord markers), the heterogenous morphology of the germ cells, and globules of basement membrane are useful differential features. The lack of a granulomatous reaction also favors DGB over germinoma. Mistaking DGB for GB with invasive germinoma may result in more aggressive therapy than warranted. The likely relationship of DGB to the relatively recently described concept of so-called "undifferentiated gonadal tissue" is discussed herein.

An Unusual Presentation of 46,XY Pure Gonadal Dysgenesis: Spontaneous Breast Development and Menstruation.

46,XY pure gonadal dysgenesis (Swyer syndrome) is characterized by normal female genitalia at birth. It usually first becomes apparent in adolescence with delayed puberty and amenorrhea. Rarely, patients can present with spontaneous breast development and/or menstruation. A fifteen-year-old girl presented to our clinic with the complaint of primary amenorrhea. On physical examination, her external genitals were completely female. Breast development and pubic hair were compatible with Tanner stage V. Hormonal evaluation revealed a hypergonadotropic state despite a normal estrogen level. Chromosome analysis revealed a 46,XY karyotype. Pelvic ultrasonography showed small gonads and a normal sized uterus for age. SRY gene expression was confirmed by multiplex polymerase chain reaction. Direct sequencing on genomic DNA did not reveal a mutation in the SRY, SF1 and WT1 genes. After the diagnosis of Swyer syndrome was made, the patient started to have spontaneous menstrual cycles and therefore failed to attend her follow-up visits. After nine months, the patient underwent diagnostic laparoscopy. Frozen examination of multiple biopsies from gonad tissues revealed gonadoblastoma. With this report, we emphasize the importance of performing karyotype analysis, which is diagnostic for Swyer syndrome, in all cases with primary or secondary amenorrhea even in the presence of normal breast development. We also suggest that normal pubertal development in patients with Swyer syndrome may be associated with the presence of a hormonally active tumor.

Gonadoblastoma and hepatoid and endometrioid-like yolk sac tumor: an update.

Dr Robert E. Scully greatly advanced our understanding of germ cell neoplasia to the extent that it is difficult to narrow the discussion of his contributions to this topic so that it can be covered in a brief article. This article accordingly focuses on some of the recent developments concerning 2 of his major contributions in this area-the gonadoblastoma (GB) and variant morphologies of yolk sac tumor. GB was defined by Dr Scully in 1953 and its features elaborated in detail by him in 1970. This neoplasm occurred in young patients who often displayed phenotypic sex ambiguities and frequently presented with primary amenorrhea. It was bilateral in 40%, and consisted of circumscribed nests of small sex cord cells and germinoma-like cells admixed with round deposits of eosinophilic, hyaline, often calcified material. These nests were set in a spindle cell gonadal stroma with Leydig-like or lutein-like cells. Because of his work we now understand that this precursor to invasive germ cell tumors occurs in patients with a specific form of disorder of sex development, namely gonadal dysgenesis, and only in those who have a particular portion of the Y chromosome, the GB locus/TSPY gene, within the gonadal tissue. An essential element to the development of GB appears to be a defect in the genetic pathway that leads to the development of Sertoli cells. Improperly formed Sertoli cells predispose to "delayed maturation" of the gonocytes of the gonad and predispose them to undergo malignant transformation. "Undifferentiated gonadal tissue" has been proposed as the precursor to the development of GB and consists of an unorganized mixture of apparently non-neoplastic germ cells, germ cells with delayed maturation, and neoplastic germ cells with sex cord cells and gonadal stroma. Two variant morphologies of yolk sac tumor were also recognized by Dr Scully. In the hepatoid variant features similar to hepatocellular carcinoma occurred, although primitive glandular foci and lack of liver involvement permitted its distinction in most cases. More recently this variant has been found to occasionally produce bile in canalicular-like structures and to stain strongly for both SALL4 and glypican 3, 2 recently described markers of yolk sac tumor. Recognition of hepatoid yolk sac tumor was followed by the description of a potential mimic, primary ovarian hepatoid carcinoma, which, however, occurred in a significantly older patient population and was occasionally associated with surface epithelial neoplasia. The endometrioid-like variant of yolk sac tumor simulated primary endometrioid adenocarcinoma. It can be suspected on routine stains because of primitive appearing nuclei, frequent subnuclear vacuoles, and in some cases association with more usual yolk sac tumor. Its recognition is now facilitated by a panel of immunohistochemical stains that are often expressed differentially in these 2 neoplasms--endometrioid-like yolk sac tumor: positive for SALL4, glypican 3, and α-fetoprotein; endometrioid adenocarcinoma: positive for cytokeratin 7 and epithelial membrane antigen. Finally, Dr Scully contributed one of the first cases in the literature of yet another nuance in the complicated world of yolk sac neoplasia, namely the development of some tumors on the background of a surface epithelial neoplasm. This is analogous to the more common development of choriocarcinoma from carcinoma and, in the case of yolk sac tumor, diagnosis is aided clinically by the usual older age of the patient and nature of the associated neoplasia.

Gonadoblastoma: an immunohistochemical study and comparison to Sertoli cell nodule with intratubular germ cell neoplasia, with pathogenetic implications.

AIMS: To investigate the immunohistochemical properties of the sex cord cells of gonadoblastoma and Sertoli cell nodule with intratubular germ cell neoplasia unclassified (IGCNU) as a means of objective distinction and to provide insight into the pathogenesis. METHODS AND RESULTS: Immunohistochemical stains for SOX9, FoxL2 and SF-1 were performed on 10 gonadoblastomas (all phenotypical females) and 14 Sertoli cell nodules with IGCNU in normal phenotypical males with coexisting germ cell tumours. The sex cord cells of gonadoblastomas showed strong, diffuse FoxL2 and SF-1 positivity and focal weak to moderate SOX9 reactivity, whereas those of Sertoli cell nodules with IGCNU were uniformly, strongly positive for SOX9 and SF-1, while negative for FoxL2. CONCLUSIONS: Coexpression of SOX9 and FoxL2 in the sex cord cells of gonadoblastomas provides evidence that these morphologically ambiguous sex cord cells are incompletely differentiated. The strong, diffuse SOX9 and SF-1 positivity and absence of FoxL2 reactivity in the Sertoli cell nodules with IGNCU support full Sertoli cell differentiation of the sex cord cells and distinguish them from gonadoblastomas. Deficient SOX9 expression in gonadoblastoma supports a current model of pathogenesis where immature germ cells, in the absence of well-formed Sertoli cells, retain a fetal phenotype and susceptibility to malignant transformation.

The Y-located gonadoblastoma gene TSPY amplifies its own expression through a positive feedback loop in prostate cancer cells.

The testis-specific protein Y-encoded (TSPY) is a repetitive gene located on the gonadoblastoma region of the Y chromosome, and has been considered to be the putative gene for this oncogenic locus on the male-only chromosome. It is expressed in spermatogonial cells and spermatocytes in normal human testis, but abundantly in gonadoblastoma, testicular germ cell tumors and a variety of somatic cancers, including melanoma, hepatocellular carcinoma and prostate cancer. Various studies suggest that TSPY accelerates cell proliferation and growth, and promotes tumorigenesis. In this report, we show that TSPY could bind directly to the chromatin/DNA at exon 1 of its own gene, and greatly enhance the transcriptional activities of the endogenous gene in the LNCaP prostate cancer cells. Domain mapping analyses of TSPY have localized the critical and sufficient domain to the SET/NAP-domain. These results suggest that TSPY could efficiently amplify its expression and oncogenic functions through a positive feedback loop, and contribute to the overall tumorigenic processes when it is expressed in various human cancers.

Expression of SALL4 and SF-1 in gonadoblastoma: useful markers in the identification of the invasive germ cell component.

Gonadoblastoma is a rare gonadal neoplasm composed of primordial germ cells and sex cord-stromal cells and usually occurs in patients with dysgenetic gonads. When patients with gonadoblastoma develop an invasive germ cell tumor, the invasive germ cell component can take the form of dysgerminoma/seminoma, embryonal carcinoma, or yolk sac tumor. In this study, we performed immunohistochemical analysis for SALL4 and steroidogenic factor-1 (SF-1) on 4 cases of gonadoblastoma to examine the expression patterns of these proteins. All of the patients were phenotypically female. One patient had Swyer syndrome, the rest had Turner syndrome. The primordial germ cell component was histologically similar to cells in dysgerminoma/seminoma in these 4 cases. Two patients showed the invasive component-dysgerminoma. As expected, SALL4 stained the germ cells and SF-1 stained the sex cord-stromal cells. There was a clear distinction between the staining patterns of these 2 cell populations. This study demonstrates the utility of SALL4 and SF-1 in determining whether or not there is an invasion in the primordial germ cell component.

Gonadoblastoma in the ovaries of a lesser galago (Galago senegalensis braccatus).

An enlarged right ovary was removed from a 14-year-old lesser galago (Galago senegalensis braccatus). Cytological preparations consisted of a heterogeneous population of neoplastic cells admixed with extracellular hyaline structures and cell-free nuclei. Microscopically, the ovary was replaced with gonadoblastoma and was composed of nests of germinal cells, including large oocyte-like cells, and sex cord-stromal cells arranged in palisading patterns around the germinal cells, the periphery of the nests and around extracellular hyaline material. The animal died 2 years after initial diagnosis. Necropsy examination revealed gonadoblastoma in the left ovary. The germinal cells of the tumour in the right and left ovaries were immunoreactive for calretinin, OCT3/4, PGP 9.5, Ki67 and/or faintly for cytokeratins. Sex cord-stromal cells were immunoreactive for calretinin, OCT3/4, GATA-4, E-cadherin and vimentin. Luteinized sex cord-stromal cells were immunoreactive for inhibin-alpha. The extracellular hyaline material was immunoreactive for laminin. This is the first case of gonadoblastoma in a non-human primate.

Foxl-2 in gonad development and pathology.

The Foxl-2 gene is involved in eyelid and ovary development. Mutations can lead to a shortened protein and malformations such as BPES associated or not to POF. Forkhead point mutation C134W is a marker of adult type granulosa cell tumors only. Foxl-2 dysregulation is also present in DSD and DSD associated tumors such as Gonadoblastoma and gonadoblastoma like intratubular undetermined germ cell neoplasia. A similar spectrum of pathology involvement is also found for WT1 and RET and gives a new insight into the relationship between development, malformations and oncogenesis.

Undifferentiated gonadal tissue, Y chromosome instability, and tumors in XY gonadal dysgenesis.

Patients with XY gonadal dysgenesis are at increased risk of developing gonadal tumors. The etiology of several cases of XY gonadal dysgenesis remains unknown, but X/XY gonadal mosaicism has been hypothesized to play a role. At the histologic level, the presence of persistent primitive sex cords containing immature germ cells in dysgenetic gonads (an entity called undifferentiated gonadal tissue, or UGT) was recently described, and these immature germ cells are thought to be at risk of neoplastic transformation. To further investigate both these aspects, we retrospectively studied the gonads from 30 patients with pure (22) and mixed (8) gonadal dysgenesis. Cytogenetic analyses performed on 35 gonads revealed that structurally abnormal Y chromosomes were lost in a majority of cells from the gonads, explaining the gonadal dysgenesis of patients bearing a rearranged Y chromosome. On the other hand, normal Y chromosomes were less often lost in gonads of patients with gonadal dysgenesis. At the histologic level, 43 of the 51 gonads presented areas characteristic of a streak; 13 of these streak gonads also presented areas of UGT. Structures resembling sex cords but without germ cells were found in many of the streaks not containing UGT, suggesting that UGT was initially present. Of the 13 gonads containing both UGT and a streak, 9 developed a tumor. The proximity of UGT with the tumors as well as the immunostaining patterns (PLAP+, OCT3/4+, and CD117/KIT+) suggests that germ cells found in UGT are a risk factor for gonadal tumors.

FOXL2 and SOX9 distinguish the lineage of the sex cord-stromal cells in gonadoblastomas.

Gonadoblastomas are mixed germ cell sex cord-stromal tumors that arise in dysgenetic gonads and are composed of immature germ cells and sex cord-stromal cells of indeterminate differentiation. FOXL2 is one of the first genes expressed in female gonad development, and it is required for proper granulosa cell differentiation during folliculogenesis. SOX9 , a downstream target of SRY , the gene in the Y chromosomal sex-determining region, is required for testicular development and for the formation and maintenance of (pre-)Sertoli cells. This study characterized the sex cord-stromal cells of gonadoblastoma by evaluating the expression of these counteracting transcription factors. Archival paraffin-embedded material of 7 gonadoblastomas, 5 of which were overgrown by dysgerminoma, was examined by immunohistochemistry for expression and localization of FOXL2 and SOX9. The sex cord-stromal cells revealed strong nuclear staining for FOXL2 and were negative for SOX9 expression. Germ cells in the gonadoblastoma and dysgerminoma components showed no FOXL2 and SOX9 expression. Areas of transition between gonadoblastoma and dysgerminoma revealed nests with a gradual reduction of FOXL2 expression. Our results support the hypothesis that the sex cord-stromal cell component of gonadoblastomas is of granulosa cell origin. In addition, FOXL2 appears to be a useful marker for the evaluation of overgrowth by dysgerminomas and for the identification of the transition zone of "dysgerminoma in situ." As FOXL2 and SOX9 are differentially expressed, they also should be useful for distinguishing gonadoblastomas from intratubular germ cell neoplasias and can help to differentiate those with a Sertoli cell component from gonadoblastoma with a granulosa cell component.

The chromosome Y-linked testis-specific protein locus TSPY1 is characteristically present in gonadoblastoma.

Gonadoblastoma is a rare gonadal neoplasm that occurs almost exclusively in individuals who are phenotypically females. Most cases develop in women who have an abnormal karyotype in which at least a portion of the centromeric region of the short arm of chromosome Y is present, a region often referred to as the GBY locus. Of the several genes present in the GBY locus, the TSPY1 gene (which encodes testis-specific protein, a protein thought to have a role in cell cycle regulation) appears to be the most likely to have a critical role in the pathogenesis of gonadoblastoma. To evaluate the association of TSPY1 with the tumor, we developed an interphase fluorescent in situ hybridization assay that uses probes that target the region of the GBY locus that contains TSPY1 and a commercially available chromosome X CEP probe. Using this set of probes in a dual-color approach, we evaluated 6 cases of gonadoblastoma identified from our files and found that both TSPY1 and chromosome X were present in 5 (84%) of 6 cases; in these 5 cases, the adjacent nonneoplastic gonadal parenchyma showed the same genotype as the tumor. Of 6 cases, 1 (16%) showed no evidence of TSPY1; in this case, which occurred in a gravida 2 para 2 woman, 2 X chromosomes were present in the nonneoplastic ovary, the gonadoblastoma, and associated dysgerminoma and granulosa cell tumors. From a basic science perspective, our data demonstrate that the TSPY1 gene is present in most gonadoblastomas, supporting the hypothesized role for TSPY1 in gonadoblastoma tumorigenesis; the lack of TSPY1 in a fertile woman suggests that other loci can, however, substitute for TSPY1 in the development of the tumor. From a clinical perspective, our data show that interphase fluorescence in situ hybridization targeting TSPY1 is a straightforward approach that can be used in the evaluation of Y-associated intersex disorders in women who develop gonadoblastoma.

Gonadoblastoma: an unusual ovarian tumor.

Gonadoblastomas are unusual benign neoplasias that frequently appear in the dysgenetic gonads of women with chromosome Y anomaly. In this study, we reviewed 3 gonadoblastoma cases, 2 of which were bilateral, in patients 21, 17, and 18 years of age. Two of them presented 46 XY karyotype and gonadal dysgenesis, whereas the third presented 46 XX karyotype. Besides, 2 of the cases were associated to dysgerminomas. In all the cases, the histologic examination showed germ cell proliferation and sex cords derivatives frequently surrounding small round deposits containing amorphous hyaline material resembling Call-Exner bodies. One of the patients died at 8 years from diagnosis because of dysgerminoma multiple metastases, one is alive with no evidence of disease at the second year of follow-up, and the evolution of the third patient remains unknown.

Gonadoblastoma locus and the TSPY gene on the human Y chromosome.

The gonadoblastoma (GBY) locus is the only oncogenic locus on the human Y chromosome. It is postulated to serve a normal function in the testis, but could exert oncogenic effects in dysgenetic gonads of individuals with intersex and/or dysfunctional testicular phenotypes. Recent studies establish the testis-specific protein Y-encoded (TSPY) gene to be the putative gene for GBY. TSPY serves normal functions in male stem germ cell proliferation and differentiation, but is ectopically expressed in early and late stages of gonadoblastomas, testicular carcinoma in situ (the premalignant precursor for all testicular germ cell tumors), seminomas, and selected nonseminomas. Aberrant TSPY expression stimulates protein synthetic activities, accelerates cell proliferation, and promotes tumorigenicity in athymic mice. TSPY binds to type B cyclins, enhances an activated cyclin B-CDK1 kinase activity, and propels a rapid G(2)/M transition in the cell cycle. TSPY also counteracts the normal functions of its X-homologue, TSPX, which also binds to cyclin B and modulates the cyclin B-CDK1 activity to insure a proper G(2)/M transition in the cell cycle. Hence, ectopic expression and actions of the Y-located TSPY gene in incompatible germ cells, such as those in dysgenetic or ovarian environments and dysfunctional testis, disrupt the normal cell cycle regulation and predispose the host cells to tumorigenesis. The contrasting properties of TSPY and TSPX suggest that somatic cancers, such as intracranial germ cell tumors, melanoma, and hepatocellular carcinoma, with detectable TSPY expression could exhibit sexual dimorphisms in the initiation and/or progression of the respective oncogenesis.

Stem cell factor as a novel diagnostic marker for early malignant germ cells.

Carcinoma in situ (CIS) of the testis is the pre-invasive stage of type II testicular germ cell tumours (TGCTs) of adolescents and adults. These tumours are the most frequently diagnosed cancer in Caucasian adolescents and young adults. In dysgenetic gonads, the precursor of type II GCTs can be either CIS or a lesion known as gonadoblastoma (GB). CIS/GB originates from a primordial germ cell (PGC)/gonocyte, ie an embryonic cell. CIS can be cured by local low-dose irradiation, with limited side effects on hormonal function. Therefore, strategies for early diagnosis of CIS are essential. Various markers are informative to diagnose CIS in adult testis by immunohistochemistry, including c-KIT, PLAP, AP-2gamma, NANOG, and POU5F1 (OCT3/4). OCT3/4 is the most informative and consistent in presence and expression level, resulting in intense nuclear staining. In the case of maturational delay of germ cells, frequently present in gonads of individuals at risk for type II (T)GCTs, use of these markers can result in overdiagnosis of malignant germ cells. This demonstrates the need for a more specific diagnostic marker to distinguish malignant germ cells from germ cells showing maturation delay. Here we report the novel finding that immunohistochemical detection of stem cell factor (SCF), the c-KIT ligand, is informative in this context. This was demonstrated in over 400 cases of normal (fetal, neonatal, infantile, and adult) and pathological gonads, as well as TGCT-derived cell lines, specifically in cases of CIS and GB. Both membrane-bound and soluble SCF were expressed, suggestive of an autocrine loop. SCF immunohistochemistry can be a valuable diagnostic tool, in addition to OCT3/4, to screen for precursor lesions of TGCTs, especially in patients with germ cell maturation delay.