RESEARCH ARTICLE
Metastatic Treated Malignant Germ Cell Tumors: Is SALL4 a Better Marker Than Placental Alkaline Phosphatase? Nicole K. Andeen, MD and Maria S. Tretiakova, MD, PhD
Abstract: Studies have shown that in the metastatic setting and after treatment, expression of immunohistochemical markers may be diminished or lost. Transcription factor SALL4 (sal-like protein 4) has been recognized as a sensitive marker for both primary and metastatic malignant germ cell tumors (MGCTs), but has not been tested in the posttreatment setting. We sought to determine the level of SALL4 expression in treatment-resistant metastatic MGCT in comparison with pan-GCT marker placental alkaline phosphatase (PLAP). Thirty-six previously treated MGCTs, 16 untreated primary testicular MGCTs, and 4 cytology specimens were immunostained for SALL4 and PLAP, and staining characteristics were evaluated. In the treated MGCT group, there was diffuse SALL4 nuclear immunoreactivity in the majority of cases (27/36, 75%), labeling seminoma, yolk-sac tumor, embryonal carcinoma, and primitive neuroectodermal components. No treated metastatic MGCT lacked SALL4 immunoreactivity. In contrast, PLAP was diffusely expressed in only 14/36 (39%) cases of treated MGCTs, showed scattered focal weak to moderate positivity in 13/36 (36%), and was virtually absent in 9/36 (25%) cases. Both markers had scattered expression limited to the epithelial components of teratomatous regions. SALL4 also outperformed PLAP on a small sample of cytology blocks. Although SALL4 is not entirely specific, it is a highly sensitive marker with strong diffuse nuclear reactivity in the majority of MGCTs in the posttreatment setting, at significantly higher levels than PLAP (P < 0.001). Persistent expression of SALL4 in metastatic MGCTs resistant to chemoradiation also raises the possibility for targeted systemic therapy as the anti-SALL4 peptide continues to be developed. Key Words: SALL4, PLAP, germ cell tumor, diagnostic marker, posttreatment (Appl Immunohistochem Mol Morphol 2016;24:210–214)
Received for publication October 13, 2014; accepted November 27, 2014. From the Department of Pathology, University of Washington, Seattle, WA. The authors declare no conflict of interest. Reprints: Nicole K. Andeen, MD, Department of Pathology, University of Washington, 1959 NE Pacific Street, P.O. Box 356100, Seattle, WA 98195 (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www. appliedimmunohist.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
210 | www.appliedimmunohist.com
A
pproximately 95% of testicular neoplasms are of germ cell origin, which have a peak incidence of 10 per 100,000 in patients between the age of 25 and 30 years.1 The incidence of testicular germ cell tumors has doubled in the last 40 years, suggesting a contribution from environmental factors including various industrial exposures and estrogens.1,2 Because of advances in chemotherapy regimens and clinical trials, malignant germ cell tumors (MGCTs) generally have a high cure rate, but recurrent or residual neoplasms require aggressive surgery and chemotherapy. On one end of the spectrum, pure seminoma accounts for 50% of malignant GCTs and 80% of these patients present at stage I with a cure rate of up to 99% with surgery and radiotherapy.2 Conversely, 40% of men with mixed nonseminoma MGCT present at stages II to IV, and receive a bleomycin, etoposide, and cisplatin chemotherapeutic regimen.2 One third of these will have residual retroperitoneal masses after treatment, which may be composed of fibrosis and entirely necrotic neoplasm, teratomatous (50% to 60%), or residual malignant (5% to 10%) components.2 In addition, about 15% of these patients will relapse after chemotherapy,2 usually within 2 years, with 3% of patients experiencing a late relapse.3 Although distinguishing MGCTs may be straightforward on routine hematoxylin and eosin examination, it may be challenging in a metastatic site and after treatment, which can also significantly alter classic morphology and diminish expression of immunohistochemical markers.4,5 For example, one study of late relapses found yolk-sac tumors with a range of unusual morphologies (including glandular, clear cell, and hepatoid), teratomas, and non-GCT malignancies including various carcinomas and sarcomas.3,6 With a large spectrum of MGCT and non-GCT morphology, discerning a MGCT relapse from a new primary may present a diagnostic challenge. Stem cell transcription factor SALL4 (sal-like protein 4), is a zinc protein essential for maintenance of embryonal stem cells, has been recently shown as a very sensitive marker for MGCTs in primary, metastatic, and primary mediastinal settings.7–10 Investigators have demonstrated strong diffuse SALL4 reactivity in seminoma, dysgerminoma, yolk sac, and embryonal carcinomas, epithelial components of teratomas, and variable positivity in mononuclear trophoblasts of choriocarcinoma, outperforming other GCT markers including placental alkaline phosphatase (PLAP), OCT3-4, CD117, CD30, alpha-feta protein, beta human chorionic gonadotropin, and glypican-3.9,11
Appl Immunohistochem Mol Morphol
Volume 24, Number 3, March 2016
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.
Appl Immunohistochem Mol Morphol
Volume 24, Number 3, March 2016
SALL4 expression has not yet been tested in MGCT after chemotherapy, irradiation, or their combination. Moreover, no studies have addressed SALL4 expression in comparison with other common pan-GCT marker PLAP. In addition, 20% to 40% of MGCTs present with metastases, and may be diagnosed on fine-needle aspirate.2,12,13 A reliable pan-GCT marker would have particular diagnostic value in settings of both limited tissue and a broad clinical and cytologic differential of metastatic malignant neoplasm of unknown primary. Therefore, we sought to determine the level of SALL4 and PLAP expression in treatment-resistant metastatic MGCT and in available cytology cases, to determine the most sensitive pan-GCT diagnostic marker in the posttreatment setting.
MATERIALS AND METHODS Case Selection The study was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. We retrospectively identified 36 cases from 34 patients with metastatic MGCTs who had been treated by chemotherapy and/or radiation therapy. Cases for the posttreatment group were specifically selected for those with viable, nonteratomatous components to test the hypothesis that SALL4 would outperform PLAP in identifying these potentially problematic diagnostic cases. Patients in this category presented at stages I to IV and had generally had 50 was chosen for a confident score of positive, due to the high background of PLAP staining and presence of admixed tumor necrosis.
RESULTS Metastatic, Previously Treated MGCTs Thirty-six cases of lymph node (n = 27) or visceral metastases (n = 9) in men with MGCTs consisted of 7 pure teratomas, 26 mixed nonseminoma germ cell tumors, and 3 pure seminomas. SALL4 and PLAP immunostaining results are summarized in Table 1 and Figures 1 and 2. There was strong SALL4 nuclear immunoreactivity in the majority of cells (mean H-score 250) in 27/36 cases (75%), labeling seminoma, yolk-sac tumor, embryonal carcinoma, and primitive neuroectodermal tumor (PNET) components. In 7/9 cases with a SALL4 H-score < 100, the predominant component was mature or immature teratoma, wherein SALL4 was only focally expressed in the epithelial component. In the other 2 cases with focal SALL4 staining (H-score
Metastatic Treated Malignant Germ Cell Tumors: Is SALL4 a Better Marker Than Placental Alkaline Phosphatase? Nicole K. Andeen, MD and Maria S. Tretiakova, MD, PhD
Abstract: Studies have shown that in the metastatic setting and after treatment, expression of immunohistochemical markers may be diminished or lost. Transcription factor SALL4 (sal-like protein 4) has been recognized as a sensitive marker for both primary and metastatic malignant germ cell tumors (MGCTs), but has not been tested in the posttreatment setting. We sought to determine the level of SALL4 expression in treatment-resistant metastatic MGCT in comparison with pan-GCT marker placental alkaline phosphatase (PLAP). Thirty-six previously treated MGCTs, 16 untreated primary testicular MGCTs, and 4 cytology specimens were immunostained for SALL4 and PLAP, and staining characteristics were evaluated. In the treated MGCT group, there was diffuse SALL4 nuclear immunoreactivity in the majority of cases (27/36, 75%), labeling seminoma, yolk-sac tumor, embryonal carcinoma, and primitive neuroectodermal components. No treated metastatic MGCT lacked SALL4 immunoreactivity. In contrast, PLAP was diffusely expressed in only 14/36 (39%) cases of treated MGCTs, showed scattered focal weak to moderate positivity in 13/36 (36%), and was virtually absent in 9/36 (25%) cases. Both markers had scattered expression limited to the epithelial components of teratomatous regions. SALL4 also outperformed PLAP on a small sample of cytology blocks. Although SALL4 is not entirely specific, it is a highly sensitive marker with strong diffuse nuclear reactivity in the majority of MGCTs in the posttreatment setting, at significantly higher levels than PLAP (P < 0.001). Persistent expression of SALL4 in metastatic MGCTs resistant to chemoradiation also raises the possibility for targeted systemic therapy as the anti-SALL4 peptide continues to be developed. Key Words: SALL4, PLAP, germ cell tumor, diagnostic marker, posttreatment (Appl Immunohistochem Mol Morphol 2016;24:210–214)
Received for publication October 13, 2014; accepted November 27, 2014. From the Department of Pathology, University of Washington, Seattle, WA. The authors declare no conflict of interest. Reprints: Nicole K. Andeen, MD, Department of Pathology, University of Washington, 1959 NE Pacific Street, P.O. Box 356100, Seattle, WA 98195 (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www. appliedimmunohist.com. Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
210 | www.appliedimmunohist.com
A
pproximately 95% of testicular neoplasms are of germ cell origin, which have a peak incidence of 10 per 100,000 in patients between the age of 25 and 30 years.1 The incidence of testicular germ cell tumors has doubled in the last 40 years, suggesting a contribution from environmental factors including various industrial exposures and estrogens.1,2 Because of advances in chemotherapy regimens and clinical trials, malignant germ cell tumors (MGCTs) generally have a high cure rate, but recurrent or residual neoplasms require aggressive surgery and chemotherapy. On one end of the spectrum, pure seminoma accounts for 50% of malignant GCTs and 80% of these patients present at stage I with a cure rate of up to 99% with surgery and radiotherapy.2 Conversely, 40% of men with mixed nonseminoma MGCT present at stages II to IV, and receive a bleomycin, etoposide, and cisplatin chemotherapeutic regimen.2 One third of these will have residual retroperitoneal masses after treatment, which may be composed of fibrosis and entirely necrotic neoplasm, teratomatous (50% to 60%), or residual malignant (5% to 10%) components.2 In addition, about 15% of these patients will relapse after chemotherapy,2 usually within 2 years, with 3% of patients experiencing a late relapse.3 Although distinguishing MGCTs may be straightforward on routine hematoxylin and eosin examination, it may be challenging in a metastatic site and after treatment, which can also significantly alter classic morphology and diminish expression of immunohistochemical markers.4,5 For example, one study of late relapses found yolk-sac tumors with a range of unusual morphologies (including glandular, clear cell, and hepatoid), teratomas, and non-GCT malignancies including various carcinomas and sarcomas.3,6 With a large spectrum of MGCT and non-GCT morphology, discerning a MGCT relapse from a new primary may present a diagnostic challenge. Stem cell transcription factor SALL4 (sal-like protein 4), is a zinc protein essential for maintenance of embryonal stem cells, has been recently shown as a very sensitive marker for MGCTs in primary, metastatic, and primary mediastinal settings.7–10 Investigators have demonstrated strong diffuse SALL4 reactivity in seminoma, dysgerminoma, yolk sac, and embryonal carcinomas, epithelial components of teratomas, and variable positivity in mononuclear trophoblasts of choriocarcinoma, outperforming other GCT markers including placental alkaline phosphatase (PLAP), OCT3-4, CD117, CD30, alpha-feta protein, beta human chorionic gonadotropin, and glypican-3.9,11
Appl Immunohistochem Mol Morphol
Volume 24, Number 3, March 2016
Copyright r 2016 Wolters Kluwer Health, Inc. All rights reserved.
Appl Immunohistochem Mol Morphol
Volume 24, Number 3, March 2016
SALL4 expression has not yet been tested in MGCT after chemotherapy, irradiation, or their combination. Moreover, no studies have addressed SALL4 expression in comparison with other common pan-GCT marker PLAP. In addition, 20% to 40% of MGCTs present with metastases, and may be diagnosed on fine-needle aspirate.2,12,13 A reliable pan-GCT marker would have particular diagnostic value in settings of both limited tissue and a broad clinical and cytologic differential of metastatic malignant neoplasm of unknown primary. Therefore, we sought to determine the level of SALL4 and PLAP expression in treatment-resistant metastatic MGCT and in available cytology cases, to determine the most sensitive pan-GCT diagnostic marker in the posttreatment setting.
MATERIALS AND METHODS Case Selection The study was approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center. We retrospectively identified 36 cases from 34 patients with metastatic MGCTs who had been treated by chemotherapy and/or radiation therapy. Cases for the posttreatment group were specifically selected for those with viable, nonteratomatous components to test the hypothesis that SALL4 would outperform PLAP in identifying these potentially problematic diagnostic cases. Patients in this category presented at stages I to IV and had generally had 50 was chosen for a confident score of positive, due to the high background of PLAP staining and presence of admixed tumor necrosis.
RESULTS Metastatic, Previously Treated MGCTs Thirty-six cases of lymph node (n = 27) or visceral metastases (n = 9) in men with MGCTs consisted of 7 pure teratomas, 26 mixed nonseminoma germ cell tumors, and 3 pure seminomas. SALL4 and PLAP immunostaining results are summarized in Table 1 and Figures 1 and 2. There was strong SALL4 nuclear immunoreactivity in the majority of cells (mean H-score 250) in 27/36 cases (75%), labeling seminoma, yolk-sac tumor, embryonal carcinoma, and primitive neuroectodermal tumor (PNET) components. In 7/9 cases with a SALL4 H-score < 100, the predominant component was mature or immature teratoma, wherein SALL4 was only focally expressed in the epithelial component. In the other 2 cases with focal SALL4 staining (H-score
