International Journal of Surgical Pathology http://ijs.sagepub.com/
SALL4 and SF-1 Are Sensitive and Specific Markers for Distinguishing Granulosa Cell Tumors From Yolk Sac Tumors Shuting Bai, Shi Wei, Amy Ziober, Yuan Yao and Zhanyong Bing INT J SURG PATHOL 2013 21: 121 originally published online 25 July 2012 DOI: 10.1177/1066896912454567 The online version of this article can be found at: http://ijs.sagepub.com/content/21/2/121
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454567 national Journal of Surgical PathologyBai et al
IJSXXX10.1177/1066896912454567Inter
SALL4 and SF-1 Are Sensitive and Specific Markers for Distinguishing Granulosa Cell Tumors From Yolk Sac Tumors
International Journal of Surgical Pathology 21(2) 121–125 © The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1066896912454567 http://ijs.sagepub.com
Shuting Bai, MD, PhD1, Shi Wei, MD, PhD2, Amy Ziober, JD1, Yuan Yao, MD1, and Zhanyong Bing, MD, PhD1
Abstract Granulosa cell tumors are classified as juvenile and adult types. They may be misinterpreted as a yolk sac tumor when they exhibit a “reticular” growth pattern and contain prominent mitotic activity. In this study, the authors performed immunohistochemical stains for SALL4 and steroidogenic factor-1 (SF-1) on 27 cases of yolk sac tumors and 24 granulosa cell tumors. Nuclear stains for both antibodies were considered as positive and the intensity of staining was graded as negative, weak, moderate, and strong. All the yolk sac tumors were positive for SALL4 (100%) with moderate to strong grade staining and negative for SF-1 (100%). In contrast, all the granulosa cell tumors were positive for SF-1 (85% moderate to strong grade staining and 15% weak staining) and negative for SALL4 (100%). The difference was significant (P < .01, Student’s t test). This result indicates that these 2 markers could be used to distinguish these 2 tumors in a difficult situation. Keywords granulosa cell tumor, yolk sac tumor, SALL4, SF-1
Introduction Granulosa cell tumors are the most common type of ovarian sex-cord stromal tumor, which have malignant potential and comprise 2% to 5% of all ovarian tumors.1,2 There are 2 subtypes—adult and juvenile. The adult subtype, which occurs most commonly in middle-aged and older women (median age 50-54 years), comprises 95% of these tumors. On the other hand, the juvenile type typically develops before puberty and is more common among children and young women and accounts for 5%.3 This subtype tends to have a higher proliferative rate than the adult type and has a lower risk for late recurrences. Granulosa cell tumor in the testicle is rare and there are only 29 cases of adult type reported in the English literature.4,5 Yolk sac tumor (also called endodermal sinus tumor) is a malignant germ cell tumor that usually arises in the gonads.6 This is the third most common form of ovarian malignant germ cell tumor (˜1% of cases) and is a common component of mixed malignant germ cell tumors of the testis.7 In the ovary, most of them occur as pure forms, whereas pure yolk sac tumors of the testis are rare in adults but is the most common testicular germ cell tumor in
children. The pure testicular yolk sac tumor in children peaks at 1.5 years of age and represents about 70% of pediatric testicular germ cell tumors.8 There are numerous growth patterns of yolk sac tumors, such as tubular, cystic, papillary, solid, glandular, reticular, and hepatoid patterns.9-14 Granulosa cell tumor, especially juvenile type, may be misinterpreted as a yolk sac tumor when it exhibits a “reticular” growth pattern and contains prominent mitotic activity.1 Even though immunohistochemical (IHC) expression of α-fetoprotein and inhibin-α can be used to distinguish these 2 tumors, the reliability of antibodies targeting α-fetoproteins in tissues is often questioned. IHC for inhibin-α appears to be the most sensitive and specific for granulosa cell tumors.15,16 Malignant germ cell tumors, including yolk sac tumors, can also be positive for this 1
Hospital of the University of Pennsylvania, Philadelphia, PA, USA University of Alabama, Birmingham, AL, USA
2
Corresponding Author: Zhanyong Bing, Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, 6 Founders, 3400 Spruce Street, Philadelphia, PA 19104, USA Email:
[email protected]
122 marker.17 Since the approaches to treating these 2 tumors are drastically different, alternative markers need to be explored. Recently, 2 novel markers were identified for aiding in the diagnosis of germ cell tumors and sex-cord stromal tumors, SALL4 and steroidogenic factor-1 (SF-1). SALL4, a zinc finger transcriptional factor, was noted as a specific marker for subtyping of germ cell tumors, including seminoma, embryonal carcinoma, and yolk sac tumor.18,19 SF-1 is a nuclear receptor that plays an important role in regulating many aspects of adrenal and reproductive development and function.20-23 SF-1 has been shown to be expressed in various sex-cord stromal tumors, including Sertoli cell tumor, Sertoli–Leydig cell tumor, steroid cell tumor and fibroma/fibrothecoma, and juvenile granulosa cell tumor.24-26 Whether SALL4 and SF-1 can be used as a concise panel of markers to distinguish yolk sac tumor from granulosa cell tumor has not been elucidated. In this study, we evaluated IHC stains for SALL4 and SF-1 in the differential diagnosis of yolk sac tumors and granulosa cell tumors.
International Journal of Surgical Pathology 21(2) Table 1. Clinical Histories of Granulosa Cell Tumors and Yolk Sac Tumors Group Male Female Age range in years (mean) Ovary Testis Other sites
Granulosa Cell Tumor
Yolk Sac Tumor
1 23 (3 juvenile type) 16-71 (50)
26 1 24-68 (35)
24 1 0
1 18 8
Table 2. Immunohistochemical Stains for Granulosa Cell Tumors and Yolk Sac Tumors Tumor Type Granulosa cell tumor Juvenile type Adult type Yolk sac tumor
SF-1; n (%) 24 (100) 3 (100) 21 (100) 0 (0)
SALL4; n (%) 0 (0) 0 (0) 0 (0) 27 (100)
Materials and Methods The Surgical Pathology databases of the Hospital of the University of Pennsylvania and University of Alabama at Birmingham Hospital were searched to identify granulosa cell tumors and yolk sac tumors. In all, 24 cases of granulosa cell tumors (3 juvenile type and 22 adult type) and 27 cases of yolk sac tumors were retrieved. The study was approved by the institutional review board, All cases were immunohistochemically stained with SF-1 and SALL4 antibodies. For SF-1 (clone N1665, R&D Systems, Inc, Minneapolis, MN), antigen retrieval was achieved by incubating slides for 20 minutes at 100°C in citrate buffer. This was followed by cooling to room temperature, blocking endogenous peroxidase for 10 minutes, and incubating with Protein Block (Envision + System, Dako, Glostrup, Denmark) for 30 minutes at room temperature. After primary antibody incubation for 45 minutes at room temperature, slides were washed and incubated with horseradish peroxidase polymer for 30 minutes, washed, visualized with 3,3′-diaminobenzidine (DAB), and counterstained with hematoxylin. For SALL4 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA), the immunostaining was performed using a mouse monoclonal antibody against SALL4 of human origin (clone EE-30) on the fully automated Leica Microsystems’ Bond-Max Immunohistochemistry system (Leica Microsystems, Bannockburn, IL). After heating tissue sections in a 70°C oven for 1 hour, they were transferred to the Bond-Max Autostainer for deparaffinization, clearing, epitope retrieval, and immunohistochemical staining. Heat-induced epitope retrieval was accomplished
using citrate buffer, pH 6.0 for 20 minutes at 100°C. Sections were incubated with the SALL4 antibody at 1:100 for 15 minutes at room temperature followed by detection of the antigen with a biotin-free polymeric horseradish peroxidase–linker antibody conjugate system (Refine Polymer, Leica Microsystems) for 8 minutes at room temperature. Blocking of endogenous peroxidase was performed by incubating tissue sections in 3.0% H2O2 for 7 minutes. Staining was visualized with 3,3′-diaminobenzidine substrate chromogen for 10 minutes, rinsed in di-H2O, and counterstained with hematoxylin. Slides were manually dehydrated through a series of graded alcohol, cleared in a series of xylene, and mounted. Nuclear stains for both antibodies were evaluated and the intensity of staining was graded as negative, weak, moderate, and strong. Statistical significance was derived using Student’s t test.
Results All the cases were reviewed and pathological diagnoses were confirmed and clinical histories are summarized in Table 1. Twenty-three out of 24 granulosa cell tumors (96%) were from female patients’ ovaries with the age ranging from 16 to 85 years (average 52 years), one testicular granulosa cell tumor (4%) was from a 28-year-old male patient. Three ovarian cases were juvenile type whereas the rest were adult type (Table 2). Most of the yolk sac tumors (Figure 1C) were from testicles (18 cases, 67%), 1 case from ovary (4%), and remaining cases were from anterior
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Figure 1. SF-1 and SALL4 stains in the granulosa cell tumors (A-C) and yolk sac tumors (D-F), magnification 200×. A and D, hematoxylin and eosin stains; B and E, SF-1 stains; C and F, SALL4 stains
mediastinal areas or other sites with metastatic tumors (8 cases, 29%) of male patients. All the granulosa cell tumors were positive for SF-1 (85% with moderate to strong nuclear staining and 15%
with weak nuclear staining; Figure 1B) and negative for SALL4 (100%; Figure 1C). In contrast, all the yolk sac tumors (Figure 1D) were positive for SALL4 (100%) with moderate to strong staining (Figure 1F) and negative for
124 SF-1 (100%; Figure 1E). The difference was significant (P < .01; Student’s t test).
Discussion Granulosa cell tumors, especially juvenile type, can exhibit “reticular” growth pattern, which may be misinterpreted as yolk sac tumors. As the approaches to treat these 2 tumors are drastically different, correct diagnosis is imperative. Though morphology alone may resolve the diagnoses for most cases, immunohistochemical staining may be required for differential diagnosis in a difficult situation. In this study, we evaluated expression patterns of 2 newly described markers, SALL4 and SF-1, in granulosa cell tumors and yolk sac tumors. SALL4 is a zinc finger transcriptional factor that shares homology to the Drosophila spalt (sal) gene and acts upstream of OCT 3/4, one of the markers for primitive germ cell tumors. Recently, SALL4 was shown to be strongly expressed in yolk sac tumors in addition to other germ cell tumors, such as gonadoblastoma, dysgerminoma, and embryonal carcinoma.18,19 It was also reported as being negative in granulosa cell tumors in one study (10 cases of adult type and 8 cases of juvenile type).18 SF-1, also known as Ad4-binding protein (Ad4BP), originally described in 1992, is an orphan member of the nuclear hormone receptor superfamily and is considered to play an important role in the differentiation of steroidogenic tissues. SF-1 is a nuclear transcription factor that regulates genes that are involved in steroidogenesis, development of gonads and adrenal glands, sexual differentiation, reproduction, and metabolism. In a prior study, all primary ovarian epithelial tumors and metastatic carcinomas involving the ovaries (stomach and breast) were negative for SF-1 staining.27 In this study, all granulosa cell tumors were positive for this marker, which was consistent with previous report.24 Recently, one study reported that SF-1 could be expressed in some yolk sac tumors.27 It showed that yolk sac tumors heterogeneously expressed GATA-4, GATA-6, and their target gene SF-1, whereas they remained negative for the other GATA target genes, such as anti-Mullerian hormone and inhibin-α. To further clarify the staining patterns of SALL4 and SF-1 in granulosa cell tumors and yolk sac tumors and to evaluate whether the duo could be a good concise discriminatory panel in a difficult situation for the differential diagnosis of yolk sac tumorss and granulosa cell tumors, we performed IHC stains for SALL4 and SF-1 on these 2 types of tumors. From our results, all the yolk sac tumors were positive for SALL4 and negative for SF-1, whereas all the granulosa cell tumors were positive for SF-1 and negative for SALL4. The staining pattern of SALL4 was consistent with other prior studies, which showed that SALL4 was positive in
International Journal of Surgical Pathology 21(2) some types of germ cell tumors, including seminoma/ dysgerminoma, embryonal carcinoma, and yolk sac tumors, whereas negative for granulosa cell tumors. However, interestingly, our result indicated SF-1 was totally negative in yolk sac tumor, but positive in both adult and juvenile types of granulosa cell tumors, which was different from the results of a prior study, which showed SF-1 to be heterogeneously expressed in yolk sac tumor cells.27 The cause for such discrepancy is unknown; however, it may be because of the usage of different antibodies from different manufactories. More experiments need to be performed to compare the expression of SF-1 in yolk sac tumors by using these 2 SF-1 antibodies in one institution. In summary, we compared the expression patterns of SALL4 and SF-1 in these 2 types of tumors. Our results indicated that SALL4 and SF-1 can be used as a pair of markers in distinguishing granulosa cell tumors from yolk sac tumors, particularly when juvenile type granulosa cell tumors exhibit “reticular” growth pattern, which might be misinterpreted as a yolk sac tumor. Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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