Embryonic Stem Cell Transcription Factor Signatures in the Diagnosis of Primary and Metastatic Germ Cell Tumors

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1 ORIGINAL ARTICLE Embryonic Stem Cell Transcription Factor Signatures in the Diagnosis of Primary and Metastatic Germ Cell Tumors Sandro Santagata, MD, PhD,* Keith L. Ligon, MD, PhD,*w and Jason L. Hornick, MD, PhD* Abstract: The core embryonic stem cell transcription factors (TFs) OCT3/4 (OCT4), NANOG, and SOX2 have shared as well as nonoverlapping roles in stem cell growth and differentiation. These same TFs are also expressed in various types of human germ cell tumors (GCTs), implicating them in regulation of tumor growth and differentiation. Although NANOG and OCT3/4 are sensitive and specific markers for seminoma and embryonal carcinoma, neither factor aids in the clinically important distinction of seminomatous from nonseminomatous tumors. In contrast, expression profiling data suggest that SOX2 may help with this distinction. To determine if a panel of embryonic stem cell TFs (NANOG, OCT3/4, and SOX2) can facilitate the identification and distinction of seminomatous from nonseminomatous GCTs, we evaluated their expression by immunohistochemistry in primary testicular (n = 41) and metastatic retroperitoneal (n = 43) GCTs. Our results confirm NANOG and OCT3/4 as sensitive and specific markers for primary seminoma and embryonal carcinoma and demonstrate the novel finding that NANOG is a marker for metastatic GCTs. In addition, SOX2 is expressed in embryonal carcinoma but not pure seminoma and is therefore a useful diagnostic marker for distinguishing seminomatous and nonseminomatous GCTs. In summary, we find that the embryonic stem cell TF signature of seminoma is NANOG+, OCT3/4+, and SOX2, whereas embryonal carcinoma is NANOG+, OCT3/4+, and SOX2+, and expect these immunohistochemical profiles will facilitate the diagnosis of both primary and metastatic GCTs. Key Words: germ cell tumor, immunohistochemistry, stem cell, transcription factor, seminoma, embryonal carcinoma (Am J Surg Pathol 2007;31: ) Germ cell tumors (GCTs) account for 95% of testicular neoplasms. Most of these GCTs are aggressive and can result in early and widespread From the *Department of Pathology, Brigham and Women s Hospital, Harvard Medical School; and wdepartment of Pediatric Oncology, Dana-Farber Cancer Institute; Boston, MA. Supported in part by a Stanley L. Robbins Memorial Research Fund Award to Sandro Santagata. Reprints: Jason L. Hornick, MD, PhD, Department of Pathology, Brigham and Women s Hospital, 75 Francis Street, Boston, MA ( jhornick@partners.org). Copyright r 2007 by Lippincott Williams & Wilkins metastases, although with appropriate therapeutic management, most are curable. 7 The proper identification and classification of these tumors as seminomatous or nonseminomatous is therefore critical both for determining prognosis and for selecting treatment. The evaluation of GCTs is generally straightforward in orchiectomy specimens owing to the classic morphology exhibited by the various germ cell elements and the large size and integrity of the resection samples. Classification of metastatic GCTs in the retroperitoneum, however, can be challenging, particularly in the setting of small sample size, extensive necrosis, crush artifact, loss of morphologic features after the treatment, and dense lymphocytic or granulomatous infiltrates obscuring the tumor cells. 26,35 The diagnosis can be further complicated in the setting of an occult testicular primary, 20 or after the development of a second malignancy following treatment. 30 Compounding these diagnostic difficulties, particularly in retroperitoneal lymph node dissections, greater than 60% of GCTs are composed of a mixture of 2 or more histologic patterns. 35 Several immunohistochemical markers have been traditionally used to facilitate the diagnosis of GCTs, both primary and metastatic. These markers include placental alkaline phosphatase (PLAP), CD117 (c-kit), and CD30. 27,32,34 PLAP is expressed in both seminoma and embryonal carcinoma, whereas c-kit is often positive in seminoma, and CD30 in embryonal carcinoma. 6,11,16 These markers are limited, however, owing to relatively modest sensitivity and specificity. In addition, PLAP frequently demonstrates high background staining 10,23,26 that can be particularly challenging in retroperitoneal lymph node resections, which often show extensive necrosis. Most recently, OCT3/4 (OCT4), NANOG, and SOX2 have been identified as core transcription factors (TFs) involved in the maintenance of pluripotency and self-renewal in embryonic stem cells. 2,3,15,19,24 Studies of OCT3/4 and NANOG have shown these TFs to be highly sensitive and specific markers for seminoma/germinoma and embryonal carcinoma in primary testicular and central nervous system (CNS) tumors. 9,10,12,13,23,28 The utility of OCT3/4 has also been demonstrated in the diagnosis of metastatic seminoma and embryonal carcinoma. 5,26 Although useful for identifying GCTs, OCT3/4 and NANOG do not help with the important distinction between seminomatous and nonseminomatous tumors. 836 Am J Surg Pathol Volume 31, Number 6, June 2007

2 Am J Surg Pathol Volume 31, Number 6, June 2007 Stem Cell TF Signatures in GCTs Recent studies using gene profiling methodologies and immunohistochemical validation in individual tumor samples have suggested that high level expression of SOX2 may play a role in differentiating seminomatous and nonseminomatous GCTs of the testis, 14,25 as well as in distinguishing germinomatous and nongerminomatous CNS GCTs. 23 The diagnostic utility of SOX2 protein expression in the distinction between embryonal carcinoma and seminoma/germinoma, however, has not yet been evaluated using immunohistochemistry in a large cohort of clinically relevant GCT samples. In this study, we explore the expression of the embryonic stem cell TFs OCT3/4, NANOG, and SOX2 in primary and metastatic GCTs. We demonstrate for the first time that NANOG is both a highly sensitive and specific marker of seminoma and embryonal carcinoma in the metastatic setting, and is therefore useful, along with OCT3/4, in the diagnostic work-up of tumors metastatic to the retroperitoneum. In addition, we show that the expression of SOX2 is highly reliable in distinguishing embryonal carcinoma from seminoma in both primary testicular GCTs and retroperitoneal metastases that concomitantly express NANOG and OCT3/4. Our findings demonstrate that the core transcription regulatory factors of embryonic stem cells provide a robust and effective immunohistochemical TF signature for the diagnosis of embryonal carcinoma (NANOG+, OCT3/ 4+, and SOX2+), which can be readily distinguished from the characteristic signature of seminoma (NA- NOG+, OCT3/4+, and SOX2 ). MATERIALS AND METHODS Tissue Samples Cases were retrieved from the surgical pathology files of the Brigham and Women s Hospital, Boston, MA, in accordance with the rules and regulations stipulated by the institutional review board. Representative hematoxylin and eosin (H&E)-stained tissue sections of each case were reviewed by 2 of the authors (S.S. and J.L.H.) in all cases to confirm the diagnoses. GCT diagnoses were based on World Health Organization 35 criteria. All of the specimens were obtained as surgical resection specimens. Primary testicular GCTs were all stained with NANOG, OCT3/4, and SOX2 and included 21 cases of pure GCTs (14 seminoma, 3 embryonal carcinoma, and 4 teratoma) and 20 cases of mixed GCTs (including 15 foci of embryonal carcinoma, 6 seminoma, 15 teratoma, 6 yolk sac tumor, and 5 choriocarcinoma). Retroperitoneal metastatic GCTs were all stained with NANOG, OCT3/ 4, and SOX2 and included 43 cases with components of seminoma (n = 8), embryonal carcinoma (n = 21), teratoma (n = 16), yolk sac tumor (n = 6), and choriocarcinoma (n = 2). Eighty-five non-gcts were stained with NANOG and included 35 metastatic carcinomas (5 from colon, 3 pancreas, 5 prostate, 5 kidney, 3 stomach, 5 nonsmall cell lung, 9 carcinoma of unknown primary), 10 metastatic melanoma, 7 epithelioid mesothelioma, 5 retroperitoneal leiomyosarcoma, 7 gastrointestinal stromal tumors, 6 retroperitoneal dedifferentiated liposarcoma, 10 retroperitoneal classical Hodgkin lymphoma, and 5 retroperitoneal diffuse large B-cell lymphoma. Slide Preparation, Immunohistochemistry, and Scoring All specimens were fixed in 10% buffered formalin. Four-micron sections were cut from paraffin blocks and slides were stained with H&E. Immunohistochemical studies were performed on serial sections of the paraffin blocks. The antigens, clones, dilutions, pretreatment/ antigen retrieval conditions, and vendors of the primary antibodies are listed in Table 1. Positive and negative controls were used as appropriately indicated. The Envision Plus detection system (Dako, Carpinteria, CA) was used for visualization of all of the antibodies. For NANOG, OCT3/4, and SOX2, nuclear staining was considered a positive result. Semiquantitative grading of immunoreactivity was performed as follows: 0, no tumor cells staining; 1+, <5% of tumor cells showing reactivity; 2+, 5% to 25% of tumor cells; 3+, >25% to 50% of tumor cells; 4+, >50% to 75% of tumor cells, and 5+, >75% to 100% of tumor cells. RESULTS Stem Cell TF Immunohistochemical Signatures Are Sensitive and Specific in Differentiating Primary Seminoma and Embryonal Carcinoma Because SOX2 shows relative over-expression in embryonal carcinoma compared with seminoma/germinoma by gene expression profiling of testicular tumors 14 and by immunohistochemistry in CNS tumors, 23 we asked whether the core stem cell TFs NANOG, OCT3/4, and SOX2 could provide an immunohistochemical protein signature that would reliably differentiate the various types of primary testicular GCTs. The components of the mixed GCTs evaluated and the immunohistochemical staining results for each component are provided in Table 2, and the results for all primary testicular GCTs are summarized in Table 3. NANOG and OCT3/4 were widely expressed in all primary GCTs with seminoma (20 of 20 cases; 100%) (Figs. 1A, B) and TABLE 1. Antibody Panel Used in This Study Antigen Clone Dilution Antigen Retrieval Vendor NANOG Polyclonal 1:1000 Microwave; citrate buffer R&D Systems, Minneapolis, MN OCT3/4 C-10 1:2000 Steamer; citrate buffer Santa Cruz Biotechnology, Santa Cruz, CA SOX2 Polyclonal 1:4000 Microwave; citrate buffer Chemicon International, Temecula, CA r 2007 Lippincott Williams & Wilkins 837

3 Santagata et al Am J Surg Pathol Volume 31, Number 6, June 2007 TABLE 2. Testicular Mixed GCTs: Components and Immunostaining Results Case Embryonal Carcinoma (%) N O S Seminoma (%) N O S Teratoma (%) N O S Yolk Sac Tumor (%) N O S Choriocarcinoma (%) N O S indicates no staining; 1+, <5% tumor cells reactive; 2+, 5% to 25% tumor cells reactive; 3+, 26% to 50% tumor cells reactive; 4+, 51% to 75% tumor cells reactive; 5+, >75% tumor cells reactive. N indicates NANOG; O, OCT3/4; S, SOX2. embryonal carcinoma components (18 of 18 cases; 100%) (Figs. 1D, E) but were not expressed in teratomas (0 of 19 cases; 0%) (Figs. 1G, H), yolk sac tumors (0 of 6 cases; 0%) (Figs. 1J, K), or choriocarcinomas (0 of 5 cases; 0%) (Figs. 1M, N). Interestingly, diffuse nuclear expression of SOX2 was present in all cases of embryonal carcinoma (18 of 18 cases; 100%) (Fig. 1F). Seventeen of the 18 cases (94.4%) showed 5+ strong nuclear SOX2 staining, whereas the remaining case (1 of 18; 5.6%) showed 4+ strong nuclear staining. Scattered SOX2-positive cells were identified in one case of seminoma (1 of 20 cases; 5.0%), whereas the remaining seminomas were completely negative for SOX2 (Fig. 1C). In the single positive case, less than 1% of the tumor cells showed SOX2 nuclear reactivity (Fig. 2). Expression of SOX2 was also observed to varying degrees in the epithelium of teratomas (Fig. 1I) in 17 of 19 cases (89.5%). Five cases showed 1+ staining, 5 showed 2+, 1 showed 3+, and 6 showed 4+ staining. None of the teratomas showed 5+ nuclear reactivity, and only one tumor showed focal (2+) reactivity in stromal elements. None of the yolk sac tumors (0 of 6 cases; 0%) (Fig. 1L) or choriocarcinomas (0 of 5 cases; 0%) (Fig. 1O) expressed SOX2. Figure 3 illustrates OCT3/4, NANOG, and SOX2 staining in adjacent components of a testicular mixed GCT. NANOG Is a Sensitive and Specific Marker for Metastatic Retroperitoneal Seminoma and Embryonal Carcinoma We evaluated immunohistochemical staining for NANOG in a large cohort of metastatic GCTs to the retroperitoneum. The immunohistochemical staining results are summarized in Table 4. Interestingly, NANOG was expressed in the nuclei of tumor cells in all foci of TABLE 3. Summary of Immunostaining Results for Primary Testicular GCTs No. Positive Cases (%) Tumor NANOG OCT3/4 SOX2 Total No. Cases Pure seminoma 14 (100%) 14 (100%) 0 (0%) 14 Pure embryonal carcinoma 3 (100%) 3 (100%) 3 (100%) 3 Pure teratoma 0 (0%) 0 (0%) 3 (75%) 4 Mixed GCT foci of 20 Embryonal carcinoma 15 (100%) 15 (100%) 15 (100%) 15 Seminoma 6 (100%) 6 (100%) 0 (0%)* 6 Teratoma 0 (0%) 0 (0%) 14 (93%) 15 Yolk sac tumor 0 (0%) 0 (0%) 0 (0%) 6 Choriocarcinoma 0 (0%) 0 (0%) 0 (0%) 5 *<1% of tumor cells showed nuclear reactivity for SOX2 in 1 case (Fig. 2). 838 r 2007 Lippincott Williams & Wilkins

4 Am J Surg Pathol Volume 31, Number 6, June 2007 Stem Cell TF Signatures in GCTs H&E NANOG SOX2 A B C CHORIO- CARCINOMA YOLK SAC TUMOR TERATOMA EMBRYONAL CARCINOMA SEMINOMA D E F G H I J K L M N O FIGURE 1. Stem cell TF immunohistochemical signatures are sensitive and specific in differentiating primary seminoma and embryonal carcinoma. Primary testicular GCTs stained with H&E and immunostained for NANOG and SOX2: seminoma (A C), embryonal carcinoma (D F), teratoma (G I), yolk sac tumor (J L), and choriocarcinoma (M O). Diffuse nuclear expression of NANOG is seen only in seminoma (B) and embryonal carcinoma (E). SOX2 is expressed diffusely in the nuclei of embryonal carcinoma (F), but not in seminoma (C). This distinct pattern of nuclear protein expression provides a sensitive and specific immunohistochemical expression signature to differentiate seminoma (NANOG+, OCT3/4+, and SOX2 ) from embryonal carcinoma (NANOG+, OCT3/4+, and SOX2+). SOX2 expression is often detected in the epithelium of teratomas (I), but teratomas are negative for NANOG (H) and OCT3/4. Original magnification 400. r 2007 Lippincott Williams & Wilkins 839

5 Santagata et al Am J Surg Pathol Volume 31, Number 6, June 2007 FIGURE 2. SOX2 expression is rare in seminomas. A single case of primary testicular seminoma (A; H&E) showed nuclear immunoreactivity for SOX2 (B) in rare tumor cells (<1% of cells). Diffuse nuclear expression of NANOG was seen (not shown). Original magnification 400. seminoma (8 of 8 cases; 100%) (Figs. 4A, B) and embryonal carcinoma (21 of 21 cases; 100%) (Figs. 4C, D), but was negative in all foci of teratoma (0 of 16 cases; 0%), yolk sac tumor (0 of 6 cases; 0%), and choriocarcinoma (0 of 2 cases; 0%). NANOG showed 5+ staining in all regions of seminoma. NANOG showed 4+ staining in 2 of the cases with embryonal carcinoma components, with all other 19 cases showing 5+ staining. Within the same panel of tumors, an identical pattern of staining was observed for OCT3/4 (Table 4). Moreover, to examine the specificity of NANOG for metastatic seminoma and embryonal carcinoma, a diverse panel of metastases to the retroperitoneum and other sites, many of which enter the differential diagnosis of GCTs, in addition to primary retroperitoneal sarcomas and lymphomas, were evaluated for NANOG protein expression. Nuclear expression of NANOG was absent in 83 of the 85 cases (97.6%) (Figs. 4E H). Only one case of melanoma and one metastatic carcinoma of unknown primary showed positive staining for NANOG, but these A B C D FIGURE 3. Embryonic stem cell TFs in a testicular mixed GCT. A mixed GCT containing adjacent seminoma (left) and embryonal carcinoma (right) components stained with H&E (A) and immunostained for OCT3/4 (B), NANOG (C), and SOX2 (D). Diffuse nuclear expression of OCT3/4 and NANOG is seen in both seminoma and embryonal carcinoma components, whereas expression of SOX2 is limited to embryonal carcinoma. In this case, the intensity of staining for OCT3/4 was weaker in seminoma than in embryonal carcinoma, whereas the intensity of staining for NANOG was similar in the 2 components. Original magnification r 2007 Lippincott Williams & Wilkins

6 Am J Surg Pathol Volume 31, Number 6, June 2007 Stem Cell TF Signatures in GCTs TABLE 4. Summary of Immunostaining Results for Metastatic GCTs No. Positive Cases (%) Tumor NANOG OCT3/4 SOX2 Total No. Cases Foci of Embryonal carcinoma 21 (100%) 21 (100%) 21 (100%) 21 Seminoma 8 (100%) 8 (100%) 0 (0%) 8 Teratoma 0 (0%) 0 (0%) 13 (81%) 16 Yolk sac tumor 0 (0%) 0 (0%) 0 (0%) 6 Choriocarcinoma 0 (0%) 0 (0%) 0 (0%) 2 reactive cells comprised less than 1% of the tumor cells in both cases. Stem Cell TF Immunohistochemical Signatures are Sensitive and Specific in Differentiating Metastatic Seminoma and Embryonal Carcinoma in the Retroperitoneum To further validate the stem cell TF immunohistochemical signatures of seminoma and embryonal carcinoma, we examined the expression of SOX2 (Table 4, Fig. 5) on the panel of retroperitoneal GCTs that had been previously used to test the sensitivity and specificity of NANOG as a marker for retroperitoneal metastatic seminoma and embryonal carcinoma. Similar to our findings in primary GCTs, expression of SOX2 was not observed in any seminoma (0 of 8 cases; 0%) (Fig. 5B), but was strongly and diffusely positive in the nuclei of all embryonal carcinomas (21 of 21 cases; 100%) (Fig. 5D). SOX2 showed 5+ staining in 12 of 21 cases (57.1%), 4+ in 6 of 21 cases (28.6%), and 3+ in 3 of 21 cases of metastatic embryonal carcinoma (14.3%). Expression of SOX2 was also observed to varying degrees in the epithelium of teratomas with 13 of 16 cases (81.3%) showing strong nuclear staining (Fig. 5F). Yolk sac tumors (0 of 6 cases; 0%) (Fig. 5H) and choriocarcinomas (0 of 2 cases; 0%) (Fig. 5J) did not express SOX2. These data suggest that metastatic seminoma maintains the embryonic stem cell TF signature NANOG+, OCT3/ 4+, and SOX2 and metastatic embryonal carcinoma maintains the TF profile NANOG+, OCT3/4+, and SOX2+. DISCUSSION SOX2 Is a Discriminatory Marker for Distinguishing Embryonal Carcinoma From Seminoma The histologic classification of malignant testicular GCTs, particularly the distinction between seminomatous and nonseminomatous GCTs, is important for prognosis and selection of appropriate management. Gene expression profiling has provided a lead for a robust diagnostic marker with such discriminatory potential by demonstrating that SOX2 is over-expressed in embryonal carcinoma versus seminoma. Initial exploration of this finding by immunohistochemistry was conducted in one study using single testicular seminoma and embryonal carcinoma cases, 14 and by our group using a small number of CNS mixed GCTs with embryonal differentiation and a dozen CNS germinomas. 23 A more extensive evaluation of SOX2 expression in mixed GCTs of the CNS was not possible because of the rarity of these tumors. The findings in these initial studies suggested that SOX2 might be a useful diagnostic marker for distinguishing embryonal carcinoma from seminoma/germinoma; however, the use of SOX2 as a diagnostic immunohistochemical marker required further investigation and validation in a large clinical cohort of primary and metastatic tumors. Our study using 84 primary and metastatic GCTs demonstrates widespread SOX2 expression in 100% of the 39 tumors with embryonal differentiation and limited SOX2 expression in dispersed, scattered cells in 1 of 28 (3.6%) seminomas, with no detection of SOX2 in all other seminoma cases. In the single seminoma with very limited SOX2 expression, the scattered SOX2-positive cells might represent a population of tumor cells undergoing incipient embryonal differentiation that could not be detected by conventional H&E examination. NANOG and OCT3/4 expression was present in all cases of embryonal carcinoma and seminoma. These data support that a minimal TF panel can reliably distinguish GCTs from other neoplasms with the immunohistochemical TF signature of NANOG+, OCT3/4+, and SOX2 identifying seminoma and NANOG+, OCT3/4+, and SOX2+ identifying embryonal carcinoma. Previous work from our group and others suggests that CNS GCTs likely exhibit similar TF profiles, 23 implying that this TF signature concept might be more generalized to all GCTs at various sites. The immunohistochemical panel of NANOG, OCT3/4, and SOX2 examined in this study should prove to be of use in the diagnostic work-up of GCTs, in part owing to the robust quality of the markers and the relatively straightforward nature of evaluating nuclear reactivity versus cytoplasmic or membranous positivity. SOX2 showed strong diffuse nuclear staining in the embryonal carcinoma cells with 94% of the primary testicular cases and 57% of the retroperitoneal metastases showing reactivity in >75% of the tumor cells. Eightysix percent of the retroperitoneal metastases showed SOX2 reactivity in >50% of the tumor cell nuclei. The more restricted, but still extensive, expression of SOX2 in the metastases may be secondary to decreased viability and occasional poor preservation of the tumor cells in the retroperitoneal samples, as these specimens frequently r 2007 Lippincott Williams & Wilkins 841

7 Santagata et al Am J Surg Pathol Volume 31, Number 6, June 2007 A H&E B NANOG PROSTATE CANCER MELANOMA EMBRYONAL CARCINOMA SEMINOMA C D E F G H FIGURE 4. NANOG is a sensitive and specific marker for metastatic seminoma and embryonal carcinoma in the retroperitoneum. H&E-stained sections of seminoma (A) and embryonal carcinoma (C) metastatic to the retroperitoneum. Immunohistochemistry demonstrates strong diffuse nuclear expression of one of the core transcriptional regulators of embryonic stem cells, NANOG, in the tumor cells of both seminoma (B) and embryonal carcinoma (D). Nuclear expression of NANOG was absent in tumors frequently considered in the differential diagnosis of metastatic seminoma and embryonal carcinoma such as metastatic melanoma (E, F), and metastatic prostatic carcinoma (G, H). Original magnification 400. exhibited significant necrosis and crush artifact. The use of a panel of markers in the metastatic setting provides the added security of redundancy as both OCT3/4 and NANOG reliably identify seminoma and embryonal carcinoma, and SOX2 offers the discriminatory power of subclassification as seminoma or embryonal carcinoma. Differential Expression of Embryonic Stem Cell TFs May Define Phenotypic Properties of GCTs The relative over-expression of SOX2 in embryonal carcinoma versus seminoma may explain the difference in pluripotency between these 2 tumor subtypes and the phenotypic diversity seen in mixed malignant 842 r 2007 Lippincott Williams & Wilkins

8 Am J Surg Pathol Volume 31, Number 6, June 2007 Stem Cell TF Signatures in GCTs A H&E B SOX2 CHORIO- CARCINOMA YOLK SAC TUMOR TERATOMA EMBRYONAL CARCINOMA SEMINOMA C D E F G H I J FIGURE 5. Stem cell TF immunohistochemical signatures are sensitive and specific in identifying GCTs and differentiating metastatic seminoma and embryonal carcinoma in the retroperitoneum. Metastatic GCTs in the retroperitoneum stained with H&E and immunostained for SOX2: seminoma (A, B), embryonal carcinoma (C, D), teratoma (E, F), yolk sac tumor (G, H), and choriocarcinoma (I, J). SOX2 is expressed in embryonal carcinoma (D), but not in seminoma (B). As in the primary testicular GCTs (Fig. 1), this distinct pattern of protein expression reveals a sensitive and specific immunohistochemical profile to differentiate seminoma (NANOG+, OCT3/4+, and SOX2 ) from embryonal carcinoma (NANOG+, OCT3/4+, and SOX2+). As in the primary testicular GCTs, SOX2 expression is detected in the epithelium of teratomas (F), but teratomas are negative for NANOG and OCT3/4. Original magnification 400. r 2007 Lippincott Williams & Wilkins 843

9 Santagata et al Am J Surg Pathol Volume 31, Number 6, June 2007 nonseminomatous GCTs. A recent study demonstrated that SOX2, when expressed with OCT3/4, KLF4, and c-myc, can participate in the reprogramming of differentiated cells into an embryonic stem cell-like state exhibiting the morphology and growth properties of embryonic stem cells. 29 These reprogrammed cells can form teratomas when transferred into nude mice and can contribute to mouse embryonic development when introduced into blastocysts. NANOG, interestingly, was not required for the induction of this reprogramming phenomenon. These data suggest that the expression of SOX2 in embryonal carcinoma in the presence of OCT4 and NANOG may explain why embryonal carcinoma is frequently a component of mixed nonseminomatous GCTs along with teratomatous elements that differentiate along lineages in a manner similar to that occurring during early embryogenesis. The restricted differentiation of seminoma and the frequency with which it occurs as a pure tumor rather than as a component of mixed tumors may result from the active restriction of SOX2 expression despite the presence of other embryonic stem cell TFs. NANOG Is a Sensitive and Specific Marker of GCTs in the Metastatic Setting Although the expression of NANOG had previously been demonstrated in primary seminoma and embryonal carcinoma, our study is the first to investigate the role of NANOG in the diagnosis of metastatic GCTs. Our data demonstrate that NANOG is extensively expressed in all primary and metastatic seminoma and embryonal carcinoma cases and establish NANOG as a sensitive and specific marker that can be of diagnostic utility in difficult diagnostic settings. The presence of scattered NANOG-positive cells in 2 of 85 non-gct metastases (1 melanoma and 1 carcinoma) may represent a subpopulation of tumor progenitor cells. The widespread expression of NANOG in all of the seminoma and embryonal carcinoma samples, however, was readily differentiated from the limited expression noted in these 2 cases. Hence, the limited staining pattern seen in occasional non-gcts should not pose a diagnostic pitfall. TFs as Tools in Diagnostic Surgical Pathology By establishing the global transcriptional repertoire of a cell, TFs can be primary determinants of cell lineage. During malignant transformation, expression of a subset of these lineage-identifying factors is often maintained. Lineage-specific genes, in particular TFs like NANOG and OCT3/4, can therefore serve as important diagnostic markers in surgical pathology to help identify primary tumors and predict the likely site of origin of metastatic tumors. In addition to the germ cell TFs, several other lineage-specific transcriptional regulators have found diagnostic applications in surgical pathology. These TFs include OLIG2 in the diagnosis of CNS gliomas, 18,21 CDX2 for tumors of the gastrointestinal tract, 33 OCT2 and PAX5 (BSAP) for defining neoplasms of B-cell lineage, 22 the myogenic nuclear regulatory proteins myod and myogenin (MYF4) in rhabdomyosarcoma, 4 and TTF-1 in the diagnosis of lung cancer and thyroid tumors. 1 This emergent group of diagnostic markers highlights the potential for a rational methodology for the discovery and implementation of cell-lineage defining TFs as important tools in the differential diagnosis of neoplastic lesions. Although lineage-specific TFs may be particularly useful in the classification of tumors, other TFs showing broad expression patterns, as highlighted by SOX2 in this study, may also facilitate the distinction between tumor types. SOX2, while expressed in embryonic stem cells, is not lineage-specific and has a broad expression profile in both normal and tumor tissues. 8,17 In addition to embryonal carcinoma and teratoma, SOX2 expression has been observed in lung, pancreatic, and gastric adenocarcinomas. 17,31 Nonetheless, as our data demonstrate, TFs that do not alone serve as markers of cell lineage, can be used along with cell lineage markers to define immunohistochemical profiles representative of the underlying transcriptional program and identity of various tumors. The recognition of such tumor-specific immunohistochemical signatures will provide increasing specificity in the classification of tumors and may serve to broadly enhance the armamentarium of surgical pathologists. ACKNOWLEDGMENTS The authors thank Dr Geraldine Pinkus and Ms Mei Zheng for assistance with some of the immunohistochemistry. REFERENCES 1. Bejarano PA, Baughman RP, Biddinger PW, et al. 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