Primitive Embryonic-Type Neuroectodermal/Glandular Complexes in Testicular Germ Cell Tumors: A Mimic of Embryonic-Type Neuroectodermal Tumor.

Embryonic-type neuroectodermal elements are often intimately mixed with primitive endodermal-type glands, like those of yolk sac tumors, in germ cell neoplasia in situ (GCNIS)-derived germ cell tumors of the testis. Because the primitive glands mimic tubules or rosettes of embryonic-type neuroectodermal elements, these embryonic-type neuroectodermal/glandular complexes may be misinterpreted as pure lesions of embryonic-type neuroectodermal elements, which, if of sufficient size, may lead to a diagnosis of embryonic-type neuroectodermal tumor, despite that the criteria of the World Health Organization for a "somatic-type malignancy" are not met. A diagnosis of embryonic-type neuroectodermal tumor in the testis may lead to retroperitoneal lymphadenectomy even in clinical stage I patients, and in postchemotherapy resections indicates a poor prognosis. The distinction of the neuroectodermal and glandular elements is not always straightforward based on morphology alone. We, therefore, studied 34 testis-derived germ cell tumors with embryonic-type neuroectodermal/glandular complexes and 2 purely glandular yolk sac tumors to characterize the immunophenotypes and determine an efficient immunohistochemical panel to aid in this differential. We found that GFAP, synaptophysin, and paired-like homeobox 2B (PHOX2B) expression was specific to embryonic-type neuroectodermal elements, although PHOX2B had poor sensitivity. In contrast, positive reactions with antibodies directed against AFP, villin, and CDX2 were specific for the glandular elements, although CDX2 had poor sensitivity. Other markers, including AE1/AE3 cytokeratin, SALL4, glypican 3, SOX2, SOX11, CD56, INSM1, and neurofilament, proved less helpful because of their nonspecificity and/or poor sensitivity. We conclude that the optimal immunohistochemical panel for distinguishing the components of embryonic-type neuroectodermal/glandular complexes includes stains for synaptophysin, GFAP, villin, and AFP.

Yolk Sac Differentiation in Endometrial Carcinoma: Incidence and Clinicopathologic Features of Somatically Derived Yolk Sac Tumors Versus Carcinomas With Nonspecific Immunoexpression of Yolk Sac Markers.

Endometrial somatically derived yolk sac tumors are characterized by yolk sac morphology with AFP, SALL-4, and/or Glypican-3 immunoexpression. Yolk sac marker expression, however, is not limited to tumors with overt yolk sac histology. Three hundred consecutive endometrial malignancies were assessed for immunomarkers of yolk sac differentiation. Of these, 9% expressed ≥1 yolk sac marker, including 29% of high-grade tumors. Only 3 (1%) met morphologic criteria for yolk sac differentiation; these were originally diagnosed as serous, high-grade NOS, and dedifferentiated carcinoma. Two were MMR-intact and comprised exclusively of yolk sac elements, while the dedifferentiated case was MMR deficient and had a background low-grade endometrioid carcinoma; this case also showed BRG1 loss. All 3 were INI1 intact. Nonspecific yolk sac marker expression was seen in 14 carcinosarcomas, 4 endometrioid, 2 serous, 1 clear cell, 1 dedifferentiated, 1 mixed serous/clear cell, and 1 mesonephric-like carcinoma. INI1 was intact in all cases; one showed BRG1 loss. Twenty were MMR-intact, and 4 were MMR deficient. All MMR-deficient cases with yolk sac marker expression, both with and without true yolk sac morphology, had no evidence of residual disease on follow-up, whereas 82% of MMR-intact cases developed recurrent/metastatic disease. In summary, endometrial somatically derived yolk sac tumors were rare but under-recognized. While AFP immunostaining was specific for this diagnosis, Glypican-3 and SALL-4 expression was seen in a variety of other high-grade carcinomas. INI1 loss was not associated with yolk sac morphology or immunomarker expression in the endometrium, and BRG1 loss was rare. All patients with MMR-deficient carcinomas with yolk sac immunoexpression +/- morphology were disease-free on follow-up, whereas the majority of MMR-intact cancers showed aggressive disease.

Analysis of GATA3 and FOXA2 expression suggests that downregulation of genes involved in the maintenance of a mature yolk sac tumor phenotype may underlie sarcomatoid transformation.

In the post-chemotherapy setting, germ cell tumors of the testis (GCTT) that resemble non-specific sarcomas and co-express cytokeratins and glypican-3 (GPC3) are diagnosed as "sarcomatoid yolk sac tumor postpubertal-type (YSTpt)". The diagnosis of sarcomatoid YSTpt is clinically relevant but challenging due to its rarity, non-specific histology, and negative α-fetoprotein (AFP) staining. Recently, FOXA2 has emerged as a key-gene in the reprogramming of GCTT (activating the transcription of several genes, among which GATA3), and immunohistochemical studies showed that GATA3 and FOXA2 have a higher sensitivity for non-sarcomatoid YSTpt than GPC3 and AFP. We found that sarcomatoid YSTpt did not express FOXA2 [0: 14/14 (100%)] and showed focal expression of GATA3 [0: 12/14 (85.7%), 1 + : 2/14 (14.3%)], thus suggesting that these markers are not useful in diagnosing this tumor. Furthermore, we proposed a potential mechanism of sarcomatoid transformation in the post-chemotherapy setting of GCTT, mediated by the downregulation of FOXA2 and GATA3.

Administration of a glypican-3 peptide increases the infiltration and cytotoxicity of CD8+ T cells against testicular yolk sac tumor, associated with enhancing the intratumoral cGAS/STING signaling.

BACKGROUND: Glypican-3 (GPC3) is highly expressed in testicular yolk sac tumor (TYST). GPC3 has been evaluated as a cancer vaccine for some types of tumors, but little is known on the effects of GPC3 peptide-based therapy on TYST. Here, we evaluated the antitumor effect of GPC3144-152 on TYST and its potential mechanisms. METHODS: GPC3144-152 -specific CD8+ T cells were induced by vaccine immunization and examined by ELISPOT. The CD8+ T cells were purified for testing their cytotoxicity in vitro against TYST cells by CCK-8 and TUNEL assays and in vivo against tumor growth. The influence of GPC3144-152 loading and/or cGAS silencing on the tumor growth, apoptosis and cGAS/STING signaling was tested by immunohistochemistry, immunofluorescence, flow cytometry, and Western blot. RESULTS: Vaccination with GPC3144-152 induced tumor-specific CD8+ T cells that secreted high levels of IFN-γ and granzyme B, and had potent cytotoxicity against TYST in a dose-dependent manner. Adoptive transfer of CD8+ T cells and treatment with GPC3144-152 significantly inhibited the growth of TYST tumors, but less effective for cGAS-silenced TYST tumors in vivo. Treatment with GPC3144-152 enhanced the infiltration of CD8+ T cells into the tumor environment and their cytotoxicity against TYST tumors in vivo by up-regulating granzyme B and IFN-ß expression, but down-regulating GPC3 expression in the tumors. Co-culture of CD8+ T cells with TYST in the presence of exogenous GPC3144-152 enhanced peptide-specific CD8+ T-cell cytotoxicity in vitro, accompanied by enhancing cGAS, γH2AX, TBK1, and IRF3 phosphorylation in TYST cells, but less effective in cGAS-silenced TYST cells. CONCLUSIONS: These data indicated that GPC3 peptide-specific CD8+ T cells had potent antitumor activity against TYST tumor, particularly for combined treatment with the peptide, which was partially dependent on the intratumoral cGAS/STNG signaling. GPC3 peptide vaccine may be valuable for the combination treatment of TYST.

Detailed characterization of PD-1/PD-L1 and CTLA4 expression and tumor-infiltrating lymphocytes in yolk sac tumors.

BACKGROUND: Immune checkpoint blockade (ICB) is considered as a promising approach for cancer treatment. However, the potency of ICB therapy in yolk sac tumors (YSTs) has not been confirmed, and the comprehensive analysis of tumor immune microenvironment and the expression of PD-1/PD-L1 and CTLA4 were also not thoroughly evaluated. METHODS: Immunohistochemistry was performed in formalin-fixed, paraffin-embedded tumor specimens from 23 YSTs patients to detect the density and distribution of tumor-infiltrating T cells, tertiary lymphoid structures (TLSs), as well as the expression of PD-1/PD-L1 and CTLA4. RESULTS: Overall, more than half (61 %) of all patients exhibited an immune-desert phenotype based on CD3+ T cells. PD-1 expression was identified in five tumor samples (21.7 %), and PD-L1 expression exhibited a different positive rate in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) (39.1 % and 17.4 %). Noteworthily, the rate of positive CTLA4 expression in both TCs and TILs was markedly higher (69.6 % and 56.5 %) than those of PD-1 and PD-L1 expression. Furthermore, TLSs were observed in 21.74 % of all tissues, and samples with TLSs exhibited significantly higher densities of TILs and higher expression of immune checkpoint molecules, particularly PD-1/PD-L1. In addition, tumors located in testes also exhibited a higher density of TILs and higher expression of immune checkpoint molecules. CONCLUSION: Generally a high frequency of CTLA4 expression was found, PD-1/PD-L1 expression, the immune-inflamed phenotype, and TLSs were low frequency in YSTs, however, YSTs in testes showed a higher density of TILs and higher expression of immune checkpoint molecules.

Urachal yolk sac tumor penetrating the bladder as a diagnostic challenge: a case report and review of the literature.

BACKGROUND: Yolk sac tumor (YST) is a germ cell tumor. It is primarily located in the gonads but can also occur extragonadally (extragonadal yolk sac tumor - EGYST), most commonly in the pelvis, retroperitoneum or mediastinum. Only a few YSTs of the urachus have been described. CASE REPORT: We present a rare case report of a 37-year-old male with episodes of macroscopic hematuria. The histological specimen obtained by transurethral resection showed a solid, and in some parts papillary infiltrative, high-grade tumor with numerous areas of marked nuclear atypia and clear invasion between the detrusor bundles. Glandular pattern has been observed in only minority of the tumor. Immunohistochemistry showed significant positivity for GPC3, SALL4 and cytokeratins AE1/AE3, while KRT7 and GATA3 were negative. We concluded that the biopsy findings were consistent with urothelial carcinoma with infrequent YST differentiation. In definitive surgical specimens we found a malignant epithelial, glandular and cystically arranged tumor of germinal appearance arising from urachus. The surrounding urothelium was free of invasive or in situ tumor changes. We reclassified the tumor as a urachal YST. CONCLUSION: EGYST was suspected because glandular and hepatoid structures were found, but the presence of these structures should be verified by immunohistochemistry.

Cystathionine Gamma-Lyase Is Increased in Testicular Seminomas, Embryonal, and Yolk Sac Tumors.

BACKGROUND: Testicular cancer constitutes 1.0% of male cancer and typically carries a good prognosis. As far as we are aware, the role for hydrogen sulfide in testicular cancer and the level of hydrogen sulfide-synthesizing enzyme have never been addressed. Here we examined cystathionine gamma-lyase (CSE) expression in several germ-cell testicular tumors. MATERIALS AND METHODS: Tissue microarrays were employed to examine CSE expression in 32 benign testicular samples, 88 testicular seminomas, 34 embryonal carcinomas, 4 mature teratomas, and 16 yolk sac tumors, and CSE expression was compared to that seen in benign testicular tissue. RESULTS: Compared to benign testicular tissue, CSE expression was increased in all three types of testicular neoplasm but not in mature teratomas. Highest CSE expression was identified in embryonal carcinomas, which often show a relatively aggressive clinical course. CONCLUSION: For the first time, we show that CSE is increased in several common testicular germ-cell tumor types.

Testicular Tumors: A Contemporary Update on Morphologic, Immunohistochemical and Molecular Features.

Testicular tumors are incredibly diverse and one of the most challenging areas in surgical pathology. Because of the rarity and overlapping features with numerous entities occurring in the testis and paratestis, these tumors pose a diagnostic challenge even to the most experienced general pathologists. In 2016, the latest "World Health Organization (WHO) classification of testicular tumors" was released, which incorporated several updates to the previous 2004 classification system. These updates involved several entities, including germ cell tumors, sex cord-stromal tumors, tumors containing both germ cells and sex-cord stromal cells, a miscellaneous group of testicular tumors and paratesticular tumors. In addition, significant changes were also introduced in the 2018 AJCC TNM staging (8th edition) regarding testicular tumors. The germ cell tumors are divided into 2 major groups; tumors derived from germ cell neoplasia in situ (GCNIS) and those unrelated to GCNIS. The GCNIS associated tumors include seminomatous and nonseminomatous germ cell tumors, which constitute a heterogeneous group of tumors. Non-GCNIS-associated tumors include prepubertal-type teratoma, prepubertal yolk sac tumor, mixed prepubertal-type teratoma and yolk sac tumor and spermatocytic seminoma. In the sex cord-stromal category, the tumors are classified based on their cells of origin. Most are Leydig cell tumors and Sertoli cell tumors; however, several mixed and diverse entities based on cell types are included in this group. Gonadoblastoma is the only tumor in the mixed germ cell and sex cord-stromal tumor category. Because of recent advances in molecular techniques, abundant new genetic information has emerged which helped classify the tumors based on the molecular alterations and provided insights into the tumor pathogenesis. This review focused on the updates related to testicular germ cell tumors and sex cord-stromal tumors and described the morphologic, immunohistochemical and molecular characteristics with an aim to provide a practical diagnostic approach and an update on relevant recent molecular advances.

The pioneer and differentiation factor FOXA2 is a key driver of yolk-sac tumour formation and a new biomarker for paediatric and adult yolk-sac tumours.

Yolk-sac tumours (YSTs), a germ cell tumour subtype, occur in newborns and infants as well as in young adults of age 14-44 years. In clinics, adult patients with YSTs face a poor prognosis, as these tumours are often therapy-resistant and count for many germ cell tumour related deaths. So far, the molecular and (epi)genetic mechanisms that control development of YST are far from being understood. We deciphered the molecular and (epi)genetic mechanisms regulating YST formation by meta-analysing high-throughput data of gene and microRNA expression, DNA methylation and mutational burden. We validated our findings by qRT-PCR and immunohistochemical analyses of paediatric and adult YSTs. On a molecular level, paediatric and adult YSTs were nearly indistinguishable, but were considerably different from embryonal carcinomas, the stem cell precursor of YSTs. We identified FOXA2 as a putative key driver of YST formation, subsequently inducing AFP, GPC3, APOA1/APOB, ALB and GATA3/4/6 expression. In YSTs, WNT-, BMP- and MAPK signalling-related genes were up-regulated, while pluripotency- and (primordial) germ cell-associated genes were down-regulated. Expression of FOXA2 and related key factors seems to be regulated by DNA methylation, histone methylation / acetylation and microRNAs. Additionally, our results highlight FOXA2 as a promising new biomarker for paediatric and adult YSTs.

HNF1ß is a sensitive and specific novel marker for yolk sac tumor: a tissue microarray analysis of 601 testicular germ cell tumors.

Hepatocyte Nuclear Factor 1 beta (HNF1ß) is a transcription factor which plays an important role during early organogenesis, especially of the pancreato-biliary and urogenital tract. Furthermore, HNF1ß is an established marker in the differential diagnosis of ovarian cancer and shows a distinct nuclear expression in the clear cell carcinoma subtype. Recently, it has been described in yolk sac tumor, which represents a common component in many non-seminomatous germ cell tumors. Due to its broad histologic diversity, the diagnosis may be challenging and additional tools are very helpful in the workup of germ cell tumors. Immunohistochemistry was used to study HNF1ß expression in a tissue microarray (TMA) of 601 testicular germ cell tumors including seminoma, embryonal carcinoma, yolk sac tumor, choriocarcinoma, teratoma, germ cell neoplasia in situ (GCNIS), and normal tissue. The expression pattern was compared to glypican 3 (GPC3) and α-fetoprotein (AFP), two markers currently in use for the detection of yolk sac tumor. HNF1ß showed a distinct nuclear staining in comparison to the cytoplasmic pattern of GPC3 and AFP. The sensitivity and specificity of HNF1ß were 85.4% and 96.5%, of GPC3 83.3% and 90.7%, of AFP 62.5% and 97.7%. We conclude that HNF1ß allows a reliable distinction of yolk sac tumor from other germ cell tumor components. Therefore, we propose HNF1ß as a novel and robust marker in the immunohistochemical workup of testicular germ cell tumors.

SALL4 is a useful marker for pediatric yolk sac tumors.

BACKGROUND: SALL4 is a zinc finger transcription factor that exerts its physiological role during embryo-fetal development. Analyses of SALL4 expression have shown its oncogenic role in precursor B-cell lymphoblastic lymphoma, acute and chronic myeloid leukemia, gastrointestinal, breast, and lung cancers. The aim of this study was to determine the immunohistochemical profile of SALL4 in pediatric yolk sac tumors (YSTs). METHODS AND RESULTS: Immunohistochemistry detection of SALL4 was performed in 22 cases of pediatric YSTs and 10 mature teratomas. The percentage of tumor cells stained was scored as 0, 1+ (1-30% cells), 2+ (31-60%), 3+ (61-90%), and 4+ (> 90%). To compare its sensitivity and specificity with Glypican-3 and α-fetoprotein (AFP), we also stained tumors from these cases for Glypican-3 and AFP. In contrast to AFP and glypican-3, SALL4 staining in more than 90% of the tumor cells was seen in all 22 pediatric YSTs (100% sensitivity) (P < 0.001 for both SALL4 vs. AFP and SALL4 vs. glypican-3). CONCLUSIONS: SALL4 is a sensitive marker for pediatric YSTs and it can be used to distinguish them from mature teratomas. SALL4 is likely to become a new and valuable biomarker for the diagnosis of pediatric YST.

Expression of cellular apoptosis susceptibility (CAS) in the human testis and testicular germ cell tumors.

Testicular germ cell tumors are the most frequent malignancies found in men between 15 and 44 years old. Although cellular apoptosis susceptibility (CAS) was demonstrated to be upregulated in breast cancer and colon cancer, the expression of CAS in the human testis and testicular germ cell tumors remained elusive. In the present study, CAS-positive signals were detected in the normal testicular tissues, cancer adjacent normal testicular tissues, seminoma, yolk sac tumor, and teratoma. Interestingly, the expression level of CAS in testicular germ cell tumors (TGCTs) (but not seminoma) was significantly lower than that of human testicular tissues and cancer adjacent normal testicular tissues, suggesting that decreased CAS contributed to the progression of TGCTs. Notably, the expression of CAS in seminoma was significantly higher than that of in the non-seminomas, consistent with the results from TCGA database. Furthermore, the localization of CAS is mainly restricted in the nucleus in the lesions of normal human testicular tissue and cancer adjacent normal testicular tissue. Although the expression of CAS was not significantly different between normal testicular tissue and seminoma, CAS was more enriched in cytoplasm in seminoma compared to the normal, cancer adjacent tissue and other types of TGCTs. The current results demonstrated reduced expression of CAS in the human testicular germ cell tumors and the CAS translocation from the nuclear to cytoplasm in seminoma, thereby supporting a possible role in normal testis function and in the development of seminoma.

Primary Mediastinal Yolk Sac Tumors: An Immunohistochemical Analysis of 14 Cases.

Primary mediastinal germ cell tumors are uncommon tumors that can pose diagnostic difficulties due to their morphologic spectrum and unusual site. Immunohistochemistry plays an increasing role in the diagnosis of these tumors. Whereas the immunophenotype of testicular yolk sac tumors (YST) is rather well known, the opposite is true for primary mediastinal YST leading us to investigate the immunohistochemical features of 14 such neoplasms. Fourteen cases of primary mediastinal YST were reviewed and representative whole tissue sections were selected for immunohistochemical analysis using antibodies directed against CAM5.2, SALL4, OCT3/4, glypican-3, CD30, α-fetoprotein (AFP), CD117, placental alkaline phosphatase (PLAP), GATA-3, and CDX2. The percentage of positive tumor cells and the intensity of staining were evaluated and scored. All cases (100%) showed strong and diffuse expression of CAM5.2 and SALL4, 10 cases (71%) reacted with glypican-3 and AFP in a patchy manner, 5 cases (36%) showed focal positivity with PLAP and GATA-3, 4 cases (29%) showed staining for CDX2, 3 (21%) showed expression of CD117, and a single case was positive for CD30 (7%). None of the cases showed any staining for OCT3/4. Primary mediastinal YST appear to have a similar immunohistochemical phenotype as their testicular counterparts. Coexpression of CAM5.2, SALL4, glypican-3, and AFP provides the best support for YST differentiation; however, it has to be noted that none of these markers is specific for these tumors and immunohistochemical results will always have to be interpreted in the context of morphologic, clinical, and radiologic information.

ZBTB16 is a sensitive and specific marker in detection of metastatic and extragonadal yolk sac tumour.

AIMS: Accurate histological diagnosis and classification of germ cell tumours (GCTs) is key to informing successful therapeutic and surveillance strategy. The modern therapeutic approach for yolk sac tumour (YST) is highly curative. Because YST takes on a large morphological spectrum, it can be confused for other GCT subtypes as well as somatic carcinomas, particularly when YST presents in an extragonadal or a metastatic setting. Currently available immunohistochemical markers are limited by suboptimal sensitivity and specificity. We reported recently that ZBTB16 is a sensitive and specific marker for testicular YST. ZBTB16 is absent in other GCTs and in most common somatic carcinomas, including those of gastrointestinal, pancreatobillary, respiratory, genitourinary and gynaecological tracts. The purpose of this study is to investigate the diagnostic utility of ZBTB16 in the settings of metastatic and extragonadal YST. METHODS AND RESULTS: We studied 32 archived metastatic and four extragonadal primary YSTs as well as 51 somatic malignancies for their immunohistochemical expression of ZBTB16. For comparison, α-fetoprotein (AFP) and glypican-3 were also studied in parallel. Our results demonstrated an overall sensitivity of 91.6% for ZBTB16 in detecting metastatic and extragonadal YSTs. The non-YST elements (teratoma and embryonal carcinoma) in 15 YST-containing metastatic mixed GCTs were non-reactive. With the exception of occasional myoepithelial cells of salivary gland carcinoma, all the 51 somatic malignancies were negative for ZBTB16. CONCLUSIONS: ZBTB16 is a sensitive and specific marker for YST and is diagnostically superior to AFP and glypican-3 in metastatic and extragonadal settings.

Morphoproteomics Identifies the EZH2 and SIRT1 Pathways as Potential Blocks to Differentiation in Yolk Sac Tumor of the Ovary and Provides Therapeutic Options: a Case Study.

Yolk sac tumor of the ovary is a rare but highly malignant and aggressive germ cell tumor. The objective of this case study of an ovarian yolk sac tumor was to identify putative pathways that are known to pose a block in differentiation, both in early embryogenesis and in tumorigenesis, that might be amenable to low toxicity therapies designed to promote differentiation to a more benign state and prevent recurrent disease in such tumors. The enhancer of Zeste homolog 2 (EZH2), a histone methyl transferase, and silent mating type information regulation 2 homolog 1 (SIRT1), a NAD+ histone deacetylase, are two such pathways.

Response to anti-programmed cell death protein-1 antibodies in men treated for platinum refractory germ cell cancer relapsed after high-dose chemotherapy and stem cell transplantation.

INTRODUCTION: Treatment options for patients with platinum refractory metastatic germ cell tumours (GCT) relapsing after high-dose chemotherapy and autologous stem cell transplantation are limited and survival is poor. Antibodies directed against programmed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) are currently assessed within clinical trials. We present updated data on our experience with checkpoint inhibitors as a compassionate use off-label treatment attempt for highly-pretreated patients with GCT and provide an overview of the current literature on PD-L1 expression in this rare tumour entity. PATIENTS AND METHODS: We analysed all patients with platinum refractory GCT treated with checkpoint inhibitors at our institutions between 2015 and 2017. Data were retrieved retrospectively from the patient charts. RESULTS: Seven patients were treated with nivolumab or pembrolizumab. Four patients received single-dose treatment and died shortly afterwards due to tumour progression; the remaining three patients received treatment for at least 6 months. No significant treatment toxicity was observed. Long-term tumour response was achieved in two of the three patients, both of them highly positive for PD-L1 staining. INTERPRETATION: We consider checkpoint inhibition to be efficient in carefully selected patients with platinum refractory GCT. However, predictive markers associated with tumour response are not yet known and larger prospective clinical trials are warranted.