Activation of trkA induces differentiation and inhibits the growth of JK-GMS Askin tumor cells

Peripheral primitive neuroectodermal tumor (PNET) and Ewing's sarcoma (ES) constitute a unique group of small round cell tumors in childhood and young adults that are characterized by the same chromosomal translocation t(11;22)(q24;q12). Recently, the expression of neurotrophin receptors has been found in various human tumors including PNET/ES, but the functional significance of these receptor expressions has not been documented in PNET/ES. In the present study, we investigated the biologic effects of trkA neurotrophin receptor activation by nerve growth factor (NGF) in a newly established Askin tumor cell line, JK-GMS, which constitutively expresses a high level of trkA. The activation of trkA induced differentiation and inhibited the growth of JK-GMS cells, which was characteristically associated with down-regulation of c-myc and N-myc mRNA expression. NGF did not exert significant changes in two different PNET/ES cell lines, CADO-ES1 and RD-ES, which did not express detectable levels of trkA. The biologic effects mediated by NGF were abrogated by treatment of the cells with K-252a, and the treatment with brain-derived neurotrophic factor did not affect the biologic behavior of JK-GMS cells, indicating that the effects are trkA specific. The results observed were quite similar to those of neuroblastoma cells, another childhood tumor of neural crest origin. Overall findings strongly suggest that the trkA-mediated signaling pathway plays a crucial role in controlling the basic biologic properties of JK-GMS cells.     

Flt-3 and its ligand are expressed in neural crest-derived tumors and promote survival and proliferation of their cell lines.

Flt-3 ligand (FL) is a cytokine that promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors, such as stem cell factor. Previously we have demonstrated that stem cell factor and its receptor c-kit are expressed in neural crest-derived tumor cells and that a c-kit block induces their apoptosis. Here we have evaluated the expression of flt-3 and its ligand in 12 neuroectodermal tumor cell lines from neuroblastoma (NB), neuroepithelioma (NE), Ewing sarcoma (ES), and peripheral neuroectodermal tumor (PNET) and in 38 biopsies: 19 from NB and 19 from ES and PNET. RT-PCR demonstrated the expression of flt-3 and FL in all lines. Coexpression was observed in 42% of NB and in 74% of ES and PNET biopsies. Flow cytometry confirmed the presence of membrane and cytoplasmic flt-3 and membrane FL in all lines, whereas soluble FL protein was not measurable in their supernatants. Microphysiometric demonstration of acidification of the medium provided evidence of the specific response of cell lines to FL stimulation. Specific flt-3 phosphorylation after FL treatment was also demonstrated by Western blotting analysis. In cells growing in RPMI plus 1% fetal calf serum, FL revealed a significant proliferating activity, more evident in NB and NE lines (mean increase of viable cells, 73 +/- 26% after 1 day). Treatment with flt-3 antisense oligonucleotides significantly inhibited cell growth. FL also displayed an antiapoptotic activity: after a 12-hour culture in the presence of 0.1% fetal calf serum, FL caused a 50% reduction of apoptotic cells. These results provide further evidence that neuroectodermal and hematopoietic cells share common regulatory pathways, and could be of interest in the clinical management of neuroectodermal tumors.     

Subcutaneous primitive neuroectodermal tumour in the abdominal wall of a child: long-term survival after local excision

Tumours of the peripheral primitive neuroectodermal tumour/Ewing's sarcoma (PNET/ES) family can rarely occur as primary lesions within the skin and subcutis. We present a case of subcutaneous PNET within the abdominal wall of a 33-month-old child. Histologically it was a small round cell tumour that marked strongly for CD99 and displayed ultrastructual evidence of neural differentiation. The tumour was completely excised, but due to an initial misdiagnosis no adjuvant therapy was given. Despite this, the patient is well without evidence of disease 10 years and 3 months after surgery. A review of similar cases reveals not all patients are as fortunate, suggesting combination therapy, including chemotherapy, is still the best treatment for these tumours, even when small and superficial. Our report therefore highlights the importance of recognising PNET/ES in the skin and subcutis so appropriate therapy can be administered.     

Neurogenic potential of Ewing's sarcoma cells

We investigated the capacity of eight well-characterized Ewing's sarcoma cell lines to differentiate towards a neural phenotype. Ewing's sarcoma cells expressed the neuroectoderm-associated antigens such as nerve growth factor (NGF) receptor, N-CAM (6H7 and Leu-19) and Leu-7. Ewing's sarcoma cells also exhibited the potential for neural differentiation at the mRNA level; neuron-specific medium- and low-sized filament (NF-M and NF-L) expression was induced by dibutyryladenosine cyclic monophosphate. The pattern of expression of NF-L obtained by using alternative polyadenylation sites in Ewing's sarcoma cells differed from that in peripheral primitive neuroectodermal tumour (PNET) cells, and was similar to that in undifferentiated neural tissues. Furthermore, the NGF receptors detected by immunohistochemistry were found to be non-functional as assayed byc-fos induction with NGF treatment. The results indicate that Ewing's sarcoma cells maintain a primitive phenotype and have the potential to differentiate into a neural phenotype, indicating that Ewing's sarcoma is distinct from PNET.     

Role of plasma membrane localization of the scaffold protein JSAP1 during differentiation of cerebellar granule cell precursors

We previously reported that the scaffold protein c‐Jun NH₂‐terminal kinase (JNK)/stress‐activated protein kinase‐associated protein 1 (JSAP1) functions in cerebellar granule cell precursors (GCPs) to promote their cell‐cycle exit and differentiation. In this study, we used immunocytochemistry to examine the subcellular distribution of JSAP1 in proliferating cultured GCPs. We found that when stimulated with fibroblast growth factor‐2 (FGF‐2), a factor that promotes GCP differentiation through JNK and extracellular signal‐regulated kinase (ERK) signaling, JSAP1 translocated to the plasma membrane and colocalized with activated JNK and ERK. In transfected cells expressing a constitutively activated FGF receptor (FGFR), JSAP1 and the activated FGFR colocalized at the plasma membrane with not only activated but also unphosphorylated and inactive JNK and ERK. These colocalizations did not occur when a mutant JSAP1 lacking the JNK‐binding domain was substituted for wild‐type JSAP1. Biochemical analyses of transfected cells showed that activated FGFR increased JSAP1’s affinity for JNK and ERK and that JSAP1 enhanced FGFR‐induced JNK and ERK activation. Collectively, these results suggest that when stimulated by FGFR, JSAP1 translocates to the plasma membrane, where it recruits JNK and ERK and facilitates their activation, leading to the differentiation of cerebellar GCPs.     

Specific glycosaminoglycans modulate neural specification of mouse embryonic stem cells

Mouse embryonic stem (mES) cells express a low sulfated form of heparan sulfate (HS). HS chains displayed by ES cells and their progeny become more complex and more sulfated during progression from pluripotency to neuroectodermal precursors. Sulfated epitopes are important for recognition and binding of a variety of ligands including members of the fibroblast growth factor (FGF) family. We demonstrated previously that mES cells lacking HS cannot undergo neural specification but this activity can be recovered by adding soluble heparin, a highly sulfated glycosaminoglycan (GAG). Therefore, we hypothesized that soluble GAGs might be used to support neural differentiation of HS competent cells and that the mechanisms underlying this activity might provide useful information about the signaling pathways critical for loss of pluripotency and early lineage commitment. In this study, we demonstrate that specific HS/heparin polysaccharides support formation of Sox1(+) neural progenitor cells from wild-type ES cells. This effect is dependent on sulfation pattern, concentration, and length of saccharide. Using a selective inhibitor of FGF signal transduction, we show that heparin modulates signaling events regulating exit from pluripotency and commitment to primitive ectoderm and subsequently neuroectoderm. Interestingly, we were also able to demonstrate that multiple receptor tyrosine kinases were influenced by HS in this system. This suggests roles for additional factors, possibly in cell proliferation or protection from apoptosis, during the process of neural specification. Therefore, we conclude that soluble GAGs or synthetic mimics could be considered as suitable low-cost factors for addition to ES cell differentiation regimes.     

Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells

Fibroblast growth factor (FGF), transforming growth factor (TGF)/Nodal, and Insulin/insulin-like growth factor (IGF) signaling pathways are sufficient to maintain human embryonic stem cells (ESCs) and induced pluripotent stem cells in a proliferative, undifferentiated state. Here, we show that only a few FGF family members (FGF2, FGF4, FGF6, and FGF9) are able to sustain strong extracellular-signal-regulated kinase (ERK) phosphorylation and NANOG expression levels in human ESCs. Surprisingly, FGF1, which is reported to target the same set of receptors as FGF2, fails to sustain ERK phosphorylation and NANOG expression under standard culture conditions. We find that the failure of FGF1 to sustain ES is due to thermal instability of the wild-type protein, not receptor specificity, and that a mutated thermal-stable FGF1 sustains human ESCs and supports both differentiation and reprogramming protocols.     

Involvement of MAP kinases in apoptosis of macrophage treated with Trichomonas vaginalis

A primitive protozoan parasite Trichomonas vaginalis selectively activates the signal transduction pathways in macrophages (RAW264.7). This study evaluated the correlation of these signaling pathways and T. vaginalis-induced cell apoptosis. In macrophages infected with T. vaginalis, apoptosis was assessed on the basis of DNA fragmentation on agarose gel electrophoresis. Infection of macrophages with T. vaginalis induced tyrosine phosphorylation of several proteins. Infected cells with T. vaginalis were shown to associate with phosphorylation of the extracellular signal-regulated (ERK)1/2 kinase, p38, c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases on Western blot analysis. The present finding also demonstrated a link between the ERK1/2, JNK and p38 apoptotic pathways that was modulated by T. vaginalis infection.     

Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line MizhES1

Human embryonic stem (hES) cells can be maintained in a proliferative undifferentiated state in vitro by growing them on feeder layers of mouse embryonic fibroblast (MEF) cells along with basic fibroblast growth factor (bFGF/FGF-2). To understand the molecular mechanisms involved in the requirement of bFGF in human ES cells, we investigated expression of FGF receptors and intracellular signaling events in response to bFGF in human ES cell line MizhES1. On the basis of the results of RT-PCR, clear expression of FGF receptors FGFR1, FGR2, and FGFR3 was noticed. Because MAPK, PI3K, and PKC pathways are well-known pathways triggered by bFGF in other cells, these pathways were investigated after stimulation with bFGF. bFGF did not induce activation of PI3K or PKC, but induced activation of ERK (extracellular signal-regulated kinase). To monitor the consequences of ERK activation, we examined expression of the immediate early gene c-fos, one downstream target of the MEK1/ERK pathway. mRNA and protein levels of the c-fos gene were increased by bFGF. Induction of c-Fos was dependent on MEKl. Therefore, it is likely that bFGF contributes to maintenance of human ES cells, at least in part, through the MEK1/ERK pathway.     

Concise review: regulation of embryonic stem cell lineage commitment by mitogen-activated protein kinases

Embryonic stem (ES) cells can give rise, in vivo, to the ectodermal, endodermal, and mesodermal germ layers and, in vitro, can differentiate into multiple cell lineages, offering broad perspectives in regenerative medicine. Understanding the molecular mechanisms governing ES cell commitment is an essential challenge in this field. The mitogen-activated protein kinase (MAPK) pathways extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38MAPK are able to regulate ES commitment from early steps of the process to mature differentiated cells. Whereas the ERK pathway inhibits the self-renewal of ES cells, upon commitment this pathway is involved in the development of extraembryonic tissues, in early mesoderm differentiation, and in the formation of mature adipocytes; p38MAPK displays a large spectrum of action from neurons to adipocytes, and JNK is involved in both ectoderm and primitive endoderm differentiations. Furthermore, for a given pathway, several of these effects are isoform-dependent, revealing the complexity of the cellular response to activation of MAPK pathways. Regarding tissue regeneration, the potential outcome of systematic analysis of the function of different MAPKs in different ES cell differentiation programs is discussed. Disclosure of potential conflicts of interest is found at the end of this article.     

MDM2 and CDK4 gene amplification in Ewing's sarcoma

Amplification of the MDM2 gene, which maps to chromosome band 12q13 and encodes a p53-binding protein, may result in functional inactivation of p53 and has been observed in various bone and soft tissue sarcomas. Published studies have included few cases of Ewing's sarcoma (ES) or peripheral neuroectodermal tumour (PNET), a tumour group in which alterations of the p53 pathway have so far not been extensively studied. We examined two ES cell lines, RD-ES and SK-ES-1, and 30 specimens from 27 patients (24 ES, 6 PNET; 19 primary, 4 local recurrence, 7 metastasis) for MDM2 gene amplification by Southern blot analysis. All 30 clinical specimens had been confirmed to contain sufficient ES/PNET DNA by the demonstration of a rearrangement of the t(11;22)-associated EWS gene using an EWS cDNA probe on the same blots. MDM2 gene amplification was detected in 3 of 30 specimens (10 per cent), including two ES and one PNET, but in neither of the cell lines. The three cases with amplification were morphologically typical primary tumours. Two of the three cases also showed co-amplification of the CDK4 gene, which endoces a cyclin-dependent kinase and also maps to band 12q13. Clinically, all three cases had metastatic disease at diagnosis, compared with only 1 of 15 MDM2-negative cases where the primary tumour was studied. The difference was statistically significant (P=0.005), suggesting an association of MDM2 amplification with advanced stage. Further accural and multivariate analysis of ES/PNET cases with MDM2 gene amplification will be necessary to confirm the clinical significance of these findings. The results suggest that the prevalence of MDM2 gene amplification in ES/PNET is comparable to other sarcomas, and implicate dysfunction of the p53 pathway in a subset of ES/PNET. The biological significance of CDK4 co-amplification remains to be determined.     

Fibroblast growth factor 2-stimulated proliferation is lower in muscle precursor cells from old rats

In aged skeletal muscle, impairments in regrowth and regeneration may be explained by a decreased responsiveness of muscle precursor cells (MPCs) to environmental cues such as growth factors. We hypothesized that impaired responsiveness to fibroblast growth factor 2 (FGF2) in MPCs from old animals would be explained by impaired FGF2 signalling. We determined that 5-bromo-2'-deoxyuridine (BrdU) incorporation and cell number increase less in MPCs from 32- compared with 3-month-old rats. In the presence of FGF2, we demonstrated that there were age-associated differential expression patterns for FGF receptor 1 and 2 mRNAs. Measurement of downstream signalling revealed that that mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2)–extracellular signal-regulated kinase 1/2, protein kinase C and p38 were FGF2-driven pathways in MPCs. Uniquely, protein kinase C signalling was shown to play the largest role in FGF2-stimulated proliferation in MPCs. c-Jun N-terminal kinase (JNK) signalling was ruled out as an FGF2-stimulated proliferation pathway in MPCs. Inhibition of JNK had no effect on FGF2 signalling to BrdU incorporation, and FGF2 treatment was associated with increased phosphorylation of p38, which inhibits, rather than stimulates, BrdU incorporation in MPCs. Surprisingly, the commonly used vehicle, dimethyl sulphoxide, rescued proliferation in MPCs from old animals. These findings provide insight for the development of effective treatment strategies that target the age-related impairments of MPC proliferation in old skeletal muscle.     

Expression of chondrogenic potential of mouse trunk neural crest cells by FGF2 treatment.

There is a significant difference between the developmental patterns of cranial and trunk neural crest cells in the amniote. Thus, whereas cranial neural crest cells generate bone and cartilage, trunk neural crest cells do not contribute to skeletal derivatives. We examined whether mouse trunk neural crest cells can undergo chondrogenesis to analyze how the difference between the developmental patterns of cranial and trunk neural crest cells arises. Our present data demonstrate that mouse trunk neural crest cells have chondrogenic potential and that fibroblast growth factor (FGF) 2 is an inducing factor for their chondrogenesis in vitro. FGF2 altered the expression patterns of Hox9 genes and Id2, a cranial neural crest cell marker. These results suggest that environmental cues may play essential roles in generating the difference between developmental patterns of cranial and trunk neural crest cells.     

Intercellular junctions in Ewing sarcoma/primitive neuroectodermal tumor: additional evidence of epithelial differentiation.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell-cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as 'positive'. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell-cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.     

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.     

SPREDs (Sprouty Related Proteins with EVH1 Domain) promote self‐renewal and inhibit mesodermal differentiation in murine embryonic stem cells

Background : Pluripotency, self‐renewal, and differentiation are special features of embryonic stem (ES) cells, thereby providing valuable perspectives in regenerative medicine. Developmental processes require a fine‐tuned organization, mainly regulated by the well‐known JAK/STAT, PI3K/AKT, and ERK/MAPK pathways. SPREDs (Sprouty related proteins with EVH1 domain) were discovered as inhibitors of the ERK/MAPK signaling pathway, whereas nothing was known about their functions in ES cells and during early differentiation, so far. Results: We generated SPRED1 and SPRED2 overexpressing and SPRED2 knockout murine ES cells to analyze the functions of SPRED proteins in ES cells and during early differentiation. Overexpression of SPREDs increases significantly the self‐renewal and clonogenicity of murine ES cells, whereas lack of SPRED2 reduces proliferation and increases apoptosis. During early differentiation in embryoid bodies, SPREDs promote the pluripotent state and inhibit differentiation whereby mesodermal differentiation into cardiomyocytes is considerably delayed and inhibited. LIF‐ and growth factor‐stimulation revealed that SPREDs inhibit ERK/MAPK activation in murine ES cells. However, no effects were detectable on LIF‐induced activation of the JAK/STAT3, or PI3K/AKT signaling pathway by SPRED proteins. Conclusions: We show that SPREDs promote self‐renewal and inhibit mesodermal differentiation of murine ES cells by selective suppression of the ERK/MAPK signaling pathway in pluripotent cells. Developmental Dynamics 244:591–606, 2015. © 2015 Wiley Periodicals, Inc.     

Role of mitogen-activated protein kinases in activation-induced apoptosis of T cells

A member of the mitogen-activated protein (MAP) kinase family, Jun N-terminal kinase (JNK), has been implicated in regulating apoptosis in various cell types. We have investigated the requirement for another type of MAP kinase, extracellular signal-regulated protein kinase (ERK) in activation-induced cell death (AICD) of T cells. AICD is the process by which recently activated T cells undergo apoptosis when restimulated through the T-cell antigen receptor. Here we show that both JNK and ERK are activated rapidly upon T-cell receptor (TCR) ligation prior to the onset of AICD. A chemical inhibitor of ERK activation, PD 098059, inhibits ERK activation and apoptosis, while JNK activation is not inhibited. This suggests that JNK activation is not sufficient for apoptosis. TCR cross-linking induces expression of the apoptosis-inducing factor, Fas ligand (FasL), and its expression correlates with ERK activation. In addition, apoptosis induced by direct ligation of the Fas receptor by anti-Fas antibody is not associated with ERK activation and is not inhibited by PD 098059. These data suggest that ERK activation is an early event during T-cell apoptosis induced by antigen-receptor ligation, and is not involved in apoptosis per se but in the expression of FasL. MAP kinase family members may be similarly involved in inducing apoptosis signals in other cell types.     

Involvement of p38MAPK and reactive oxygen species in icariin-induced cardiomyocyte differentiation of murine embryonic stem cells in vitro

We previously reported that treatment of icariin could significantly induce cardiomyocyte differentiation of murine embryonic stem (ES) cells in vitro. In the present study, the exact activity initiated by icariin was further confirmed and the underlying molecular mechanism was investigated. We found that cardiomyocyte differentiation was efficiently stimulated only if icariin was administrated between days 5 and 8 in differentiation course, which indicated with elevated percentage of embryoid bodies (EB) and with beating areas and up- regulated expression of alpha-actinin and troponin T. Exposure of icariin triggered intracellular reactive oxygen species (ROS) generation of EBs in 3 h, which was abolished in the presence of either NADPH oxidase inhibitor DPI or antioxidant Trolox. Meanwhile, expression of NOX4, a membrane combined enzyme responsible for ROS generation, was promoted by icariin in a dose-dependent manner. Although p38MAPK (mitogen-activated protein kinase), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal protein kinase (JNK) were spontaneously activated in early differentiation, only the phosphorylation of p38MAPK was enhanced and prolonged when icariin was present, whereas both ERK and JNK showed no response to icariin treatment. Moreover, the inducible effect of icariin was blunted by SB203580, a specific inhibitor of p38MAPK. On the contrary, neither UO126 nor SP600125, the specific inhibitor of ERK and JNK, could abolish icariin-stimulated differentiation. Nuclear location of MEF2C, which played a critical role in cardiomyocyte differentiation and could be activated by p38MAPK, was stimulated after icariin exposure. Taken together, these results suggest that ROS generation and the subsequent activation of p38MAPK are essential for the inducible function of icariin on cardiomyocyte differentiation of murine embryonic stem cells in vitro.     

Primary vaginal Ewing sarcoma: case report and review of the literature

Ewing sarcoma (ES) and primitive neuroectodermal tumor (PNET) are round-cell sarcomas with varying degrees of neuroectodermal differentiation; they are considered members of the same family of tumors based on their common genetic profile. ES/PNET are very infrequent in the female genital tract, the vagina being the rarest gynecological location, with 7 cases reported to date. The authors present a case of vaginal ES/PNET with typical morphological and immunohistochemical features. EWS gene rearrangement was demonstrated by fluorescent in situ hybridization analysis. Of the 7 previously reported cases, only 2 have molecular confirmation. The age at presentation for vaginal ES/PNET is slightly higher than that for these neoplasms in bone and soft tissues, with only 1 reported patient younger than 30 years. The outcome seems to be similar to that of other superficially sited ES/PNET and more favorable than for those observed in the more typical locations. However, the currently available data are limited, and therefore, a larger number of cases is necessary to draw any firm conclusions.