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Differential Expression of SOX2 and SOX17 in Testicular Germ Cell Tumors

Daisuke Nonaka MD
DOI: http://dx.doi.org/10.1309/AJCP7MNCNBCRN8NO 731-736 First published online: 1 May 2009

Abstract

Testicular germ cell tumors (GCTs) are subclassified to seminoma and nonseminomatous GCT for the purpose of treatment and prognostication. This study examined SOX2 and SOX17 expression patterns in a total of 67 cases, including 41 pure GCTs (32 seminomas and 9 embryonal carcinomas) and 26 mixed GCTs (9 foci of seminoma, 21 of embryonal carcinoma, 17 of yolk sac tumor, 19 of teratoma, and 5 of choriocarcinoma). All seminoma components showed diffuse SOX17 nuclear expression and were negative for SOX2. All but one of the embryonal carcinomas showed diffuse SOX2 nuclear expression with the one showing a focal reaction, whereas all were negative for SOX17. SOX17 was variably expressed in all yolk sac tumor components, but SOX2 was negative. Teratomas showed variable SOX2 and SOX17 expressions in the epithelial elements. Choriocarcinomas were negative for SOX2 and SOX17. SOX2 and SOX17 expression patterns can distinguish between seminoma and embryonal carcinoma, and this distinction may be diagnostically useful.

Key Words:
  • SOX2
  • SOX17
  • Testicular
  • Germ cell tumor
  • Seminoma
  • Embryonal carcinoma
  • Yolk sac tumor

Testicular germ cell tumors are conventionally divided into seminoma and nonseminomatous germ cell tumors, and the latter category includes embryonal carcinoma, yolk sac tumor, teratoma, and choriocarcinoma. Distinction between seminoma and nonseminomatous germ cell tumor is crucial for the purpose of treatment and prognostication. Classification of germ cell tumors in orchiectomy specimens is performed by histopathologic evaluation without much difficulty in the majority of tumors; however, it can be challenging in situations such as markedly necrotic tumor and small biopsy specimens from metastatic tumor with artifact.1,2

There have been a handful of immunohistochemical studies to differentiate between the 2 categories. Recently there has been an ever-increasing interest in immunohistochemical applications of transcription factors in diagnostic pathology. Of particular interest in regard to the category of germ cell tumor is stem cell transcription factors. OCT3/4, also known as OCT4 and POU5F1, has been reported as a sensitive and specific marker for seminoma and embryonal carcinomas.35 SOX2 has been described as a useful discriminatory marker between embryonal carcinoma and seminoma, with embryonal carcinoma being positive.3,6 SOX17 has been reported as being expressed by seminoma but not by embryonal carcinoma.6,7 The present study applied SOX2 and SOX17 immunohistochemical staining in a diagnostic context to evaluate expression patterns in a variety of germ cell tumors with special interest in seminoma and embryonal carcinoma.

Materials and Methods

A total of 67 cases, including 41 pure germ cell tumors (32 seminomas and 9 embryonal carcinomas) and 26 mixed germ cell tumors (including 9 foci of seminoma, 21 of embryonal carcinoma, 17 of yolk sac tumor, 19 of teratoma, and 5 of choriocarcinoma), were retrieved. H&E-stained slides of all cases were reviewed, and the histologic diagnosis was based on recent classifications.8 Immunohistochemical analysis was performed on formalin-fixed, paraffin-embedded tissue samples using SOX2 (rabbit polyclonal, dilution 1:70, citrate buffer; Neuromics, Edina, MN) and SOX17 (goat polyclonal, dilution 1:500, citrate buffer; Neuromics) with the avidin-biotin peroxidase method in a DAKO AutoStainer (Carpinteria, CA). Appropriate positive and negative control samples were included with the study sections. The extent of nuclear staining was graded as follows: 1+, 1% to 25%; 2+, 25% to 50%; 3+, 50% to 75%; and 4+, 75% or more.

Results

The immunohistochemical results are listed in Table 1. All seminoma components from pure seminoma and seminomatous areas of mixed germ cell tumors showed 4+ SOX17 nuclear expression, whereas all seminomas were negative for SOX2 Image 1. Conversely, embryonal carcinomas expressed diffuse SOX2 nuclear expression in the majority of tumors (2+ in 1 tumor, 3+ in 1, and 4+ in 28), whereas they were completely negative for SOX17 (Image 1). The 17 yolk sac tumor components in mixed germ cell tumors showed variable SOX17 expression (1+ in 2 tumors, 2+ in 3, 3+ in 7, and 4+ in 5), but they were completely negative for SOX2 (Image 1). This reverse pattern of SOX2 and SOX17 expression was well demonstrated in the transitional area between the embryonal carcinoma and yolk sac tumor components in mixed germ cell tumors Image 2. Teratomas showed variable SOX2 and SOX17 expression (Image 1). Of 19 teratomas, 14 expressed SOX2, with 1+ in 4 tumors, 2+ in 5, 3+ in 3, and 4+ in 2. SOX2+ elements included glandular epithelium in 13 tumors, squamous epithelium in 4, neural tissue in 2, and cartilage in 2. Of the 19 teratomas, 8 also variably expressed SOX17, with 1+ in 3 tumors, 2+ in 3, 3+ in 1, and 4+ in 1, and its expression was seen only in the epithelial components. Neither SOX2 nor SOX17 was expressed in choriocarcinomas (Image 1).

View this table:
Table 1

Intratubular germ cell neoplasia, unclassified type, was seen in 27 (84%) of 32 seminomas, 9 (100%) of 9 pure embryonal carcinomas, and 22 (85%) of 26 mixed germ cell tumors, and it was highlighted by SOX17 staining with a 4+ reaction Image 3 but was negative for SOX2. Three embryonal carcinomas were associated with intratubular embryonal carcinoma, which showed a SOX17(–)/SOX2(4+) pattern. In addition, SOX17 expression was seen in the epithelial cells of the rete testis and vascular endothelial cells in diffuse staining, and in gonocytes in focal and weak reaction.

Discussion

Although thorough routine microscopic examination would allow a definitive diagnosis in the majority of germ cell tumors, a subset of tumors may require immunohistochemical staining for classification owing to unusual morphologic features, eg, a diffuse arrangement of clear cells, tumors with a glandular pattern, and tumors with a microcystic pattern.1 In the mixed germ cell tumor, one component is often intermingled intimately with others, and a small focus of a reticular or myxomatous pattern of yolk sac tumor component contiguous to embryonal carcinoma might be overlooked (Image 2).9 Immunohistochemical studies will identify such an area and allow for the quantification of each component of the mixed tumor more accurately, and it is recommended documenting the latter in the pathology report.8

The combination of CD30 and CD117 staining has been used to distinguish between embryonal carcinoma and seminoma.1,10 Approximately 90% of seminomas diffusely express CD117 in a membranous pattern,2,10,11 whereas CD117 expression is generally not seen in embryonal carcinoma, although its cytoplasmic expression has been reported in a subset of tumors in gonadal and extragonadal locations.10,12,13 Conversely, CD30 membranous expression is found in approximately 80% to 90% of embryonal carcinomas, generally in a diffuse pattern,10,12 and its expression in seminoma can be only focally seen.14,15 The CD30/CD117 staining pattern may distinguish between the 2 tumors in the majority of cases; however, a subset of tumors may not be distinguished by this combination of staining, and the characteristic membranous pattern necessary for the interpretation of the diagnosis in both entities may not be evident in a limited biopsy specimen.

Image 1

Comparison of H&E stain and SOX2 and SOX17 reactions for seminoma (x200), embryonal carcinoma (x200), yolk sac tumor (x200), teratoma (x100), and choriocarcinoma (x200). SOX17 is expressed by endothelial cells but not by choriocarcinoma cells.

Image 2

Transition from embryonal carcinoma to yolk sac tumor. A, H&E (x200). B, SOX2 reaction in embryonal carcinoma component and negative reaction in yolk sac tumor area (x200). C, SOX17 shows reversed pattern, ie, negative in embryonal carcinoma and positive in yolk sac tumor (x200).

Image 3

Intratubular germ cell neoplasia highlighted by SOX17 immunostain (x100).

In this respect, transcription factors are easier to interpret because of their distinct nuclear reaction. Transcription factors are proteins that control the first step of gene expression, and their role is to orchestrate the complex pathways of cellular growth and differentiation.16 Some of these molecules are tissue- and/or organ-specific, and they are frequently expressed at an early stage of embryogenesis in which they regulate specific differentiation pathways. These features make them particularly helpful for determining the cell lineage of tumors.

OCT3/4, also known as OCT4 and POU5F1, is a transcription factor that has been recognized as fundamental in the maintenance of pluripotentiality in embryonic stem cells and in primordial germ cells, and its expression disappears rapidly when the cells differentiate.17,18 OCT3/4 has been reported as a sensitive and specific marker for seminoma and embryonal carcinomas.3,5,6,19,20 Diffuse OCT3/4 expression is seen in all embryonal carcinomas and seminomas. The expression is also seen in intratubular germ cell neoplasia, unclassified type, but no expression is found in normal intratubular spermatogenic cells.4,19 In addition, OCT3/4 is not expressed by other types of germ cell tumors or tumors in other anatomic locations.36,19

The SOX family of transcription factors controls cell fate and differentiation in a multitude of processes, such as male differentiation, stemness, neurogenesis, and skeletogenesis, and it encompasses 20 Sox genes, which fall into 8 groups, A through H.21 Among them, SOX2 has been reported in the literature as a diagnostic marker for embryonal carcinoma.3,6 Other SOX transcription factors reported as diagnostic markers include SOX9 for mesenchymal chondrosarcoma22 and SOX10 as a panmelanocytic and schwannian marker.23 SOX2 was expressed in pure embryonal carcinomas and in the embryonal carcinoma component of mixed germ cell tumors in all cases reported in previous studies and the current series.3,6 It was also expressed in intratubular embryonal carcinoma in our study. SOX2 expression was seen only in scattered cells in 1 of 28 seminomas in a previous study,3 but its expression in seminoma was not seen in our series or in another report.6 SOX2 is, therefore, particularly useful as a discriminatory marker between embryonal carcinoma and seminoma.3,6

Recent reverse transcriptase–polymerase chain reaction studies revealed that SOX17 was amplified by seminoma but not by embryonal carcinoma.6,7 This finding was also confirmed at the protein level by immunohistochemical analysis in our study and in a previous report.6 SOX17 was completely negative in embryonal carcinoma, including pure embryonal carcinoma, the embryonal carcinoma component of mixed tumors, and intratubular embryonal carcinoma. It is interesting that SOX17 was also expressed in all yolk sac tumor components to a variable extent. This finding has not been reported in the literature. Yolk sac tumor is a tumor showing extraembryonic yolk sac differentiation, and it can display a variety of morphologic patterns.8 These patterns represent a morphologic continuum, and transition from one pattern to another and admixtures of several patterns are common.8 The solid area of yolk sac tumor can mimic seminoma and embryonal carcinoma, and embryonal carcinoma with a papillary or glandular pattern can be mistaken for a yolk sac tumor.1 SOX17 does not distinguish between seminoma and yolk sac tumor, but a positive reaction to SOX2 and OCT3/4 rules out the diagnosis of yolk sac tumor.

SOX2 and SOX17 were variably expressed in the epithelial components of 74% and 42% of teratomatous components, respectively. The epithelial components positive for SOX17 were always glandular epithelium; SOX2 was often expressed in the glandular epithelium but was less frequently expressed in the squamous epithelium. SOX17 is required for the development of the extraembryonic endoderm, which gives rise to the visceral endoderm and parietal endoderm (yolk sac), and the definitive endoderm, which gives rise to the foregut, including the respiratory and gastrointestinal tracts.2426 SOX2 is also expressed in the developing foregut endoderm, with the highest levels in the future esophagus and anterior stomach.27 SOX17 expression in yolk sac tumors, therefore, is not an unexpected finding, nor is expression of SOX2 and SOX17 in some epithelial structures in the teratoma components. The frequency of SOX2 expression in the teratomatous areas is in keeping with the findings of a previous study.3

In summary, SOX2 and SOX17 expression patterns can distinguish between seminoma and embryonal carcinoma. A panel of OCT3/4, SOX2, and SOX17 will be useful for the classification of germ cell tumors, ie, OCT3/4+/SOX2+/SOX17– for embryonal carcinoma, OCT3/4+/SOX2–/SOX17+ for seminoma, and OCT3/4–/SOX2–/SOX17+ for yolk sac tumor.

Acknowledgments

I thank Luis Chiriboga, PhD, for performing the immunohistochemical studies.

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