Immunohistochemical Evaluation of GATA-4 in Canine Testicular Tumors

Abstract

GATA-4 is a transcription factor expressed in Sertoli cells and less commonly in Leydig (interstitial) cells but not germ cells in adult human beings, cattle, pigs, and mice. We examined GATA-4 in 76 formalin-fixed, paraffin-embedded canine testicular tumors, including 21 Sertoli cell tumors (SCT), 28 Leydig (interstitial) cell tumors (LCT), 24 seminomas (GCT), and 3 mixed germ cell sex cord-stromal tumors (MGSCT). Our hypothesis was that immunohistochemistry for GATA-4 could discriminate between germ cell and sex cord-stromal tumors of the canine testis. SCTs (21/21) had strong diffuse nuclear and weak and inconsistent cytoplasmic staining. LCTs (27/28) also had strong diffuse nuclear staining and much weaker and granular cytoplasmic staining. GCTs were negative for this marker. Sex cord-stromal cells of MGSCT were also positive. These results indicate that GATA-4 is mainly expressed in sex cord-stromal tumors and not in germ cell tumors of the canine testis.

GATA-4 belongs to a family of transcription factors that regulate gene expression, differentiation, and cell proliferation in multiple tissues. ⁸ GATA-4, along with GATA-5 and GATA-6, is mainly expressed in heart, intestinal epithelium, gonads, and yolk sac endoderm, whereas GATA-1, GATA-2, and GATA-3 are mostly expressed in hematopoietic cells. ^6,12 GATA-4 is consistently expressed in human testicular sex cord–stromal tumors, particularly Sertoli cell tumors (SCT), but also in Leydig (interstitial) cell tumors (LCT) but not in seminomas (GCT) or postpuberal germinal cells. ⁶ In a recent report ¹³ that describes the immunohistochemical characterization of a canine mixed germ cell sex cord–stromal tumor (MGSCT), an antibody to GATA-4 intensely labeled neoplastic and normal Sertoli cells and normal Leydig (interstitial) cells, whereas the only normal germ cells labeled were spermatocytes. Neoplastic germ cells did not react with this antibody. Immunohistochemistry for GATA-4, with the exception of the above-mentioned case, has not been studied in the canine testis or its tumors. This communication describes the validation of an immunohistochemical test for GATA-4 in the characterization of canine testicular tumors.

Seventy-six testicular tumors, including 21 SCTs, 28 LCTs, 24 GCTs, and 3 MGSCTs, were categorized by using the current World Health Organization classification for tumors of the genital system of domestic animals. ⁵ Criteria for defining a tumor as MGSCT included the presence of intimately admixed neoplastic germ cells and Sertoli cells in tubular structures. ⁵ Malignant behavior (e.g., metastasis) was not reported for any of the tumors. The ages of the dogs ranged from 5 to 17 years. The average age for dogs with SCTs, Leydig cell tumors, and GCTs was 9.5, 11, and 12.2 years, respectively. The mean age of all the dogs in this study was 11 years. Tissues were fixed in 10% buffered neutral formalin and embedded in paraffin. A goat polyclonal anti-GATA-4 (sc-1237, Santa Cruz Biotechnology, Santa Cruz, CA) and immunoperoxidase technique were used. ¹³ Detection of this antigen is reportedly nuclear. ^2,8,13,16 Tumors were considered to express GATA-4 (positive for GATA-4) when more than 10% of the neoplastic population was positive. In large samples in which fixation was suboptimal, only well-fixed portions of the tumor (generally the outer 1 cm of the sample) were used for evaluation.

In normal testis from a young adult dog, antibody to GATA-4–labeled Sertoli cells (diffuse, nuclear) and early and late spermatids (paranuclear) (Fig. 1). Leydig (interstitial) cells had nuclear and cytoplasmic GATA-4 reactivity. An immature, normal testis from an 8-week-old puppy had strong nuclear and weaker cytoplasmic labeling of Sertoli cells (Fig. 2). The nucleus of numerous interstitial cells was also positive for GATA-4. LCTs (27/28) had strong diffuse nuclear labeling and much weaker and granular cytoplasmic labeling in most cases (Figs. 2,3). GATA-4 immunoreactivity in SCTs (21/21) was nuclear (strong and diffuse) (Figs. 4, 5). In fewer than 50% of SCTs, there was weak and diffuse cytoplasmic labeling. SCTs and interstitial cell tumors were considered positive for GATA-4 when there was labeling of their nuclei. Germ cell tumors did not react with the antibody to GATA-4. The Sertoli cell component, but not germ cells, of all MGSCTs consistently had GATA-4 expression (Fig. 6).

Fig. 1.

Normal adult testis; dog. Sertoli cells (arrows) have strongly labeled nuclei. Early and late spermatids (arrowheads) have paranuclear labeling. Spermatogonia and spermatocytes are negative. Leydig cell with nuclear labeling (long arrow). Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

Fig. 2.

Normal immature testis; dog. Strong nuclear and weaker cytoplasmic labeling in Sertoli cells of seminiferous tubules and interstitial cells (asterisks). Inset: Detail of staining in seminiferous tubule. Nuclei of Sertoli cells are strongly positive. No reactivity in nuclei of germ cells (arrowheads). Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

Fig. 3.

LCT; testis, dog. Strong nuclear labeling of most neoplastic cells. Sertoli cells of normal adjacent seminiferous tubules (asterisk) are also positive. Inset: Detail of nuclear labeling. There is weak cytoplasmic staining as well. Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

Fig. 4.

SCT, testis; dog. Intratubular growth. The majority of neoplastic cells have distinct nuclear labeling. Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

Fig. 5.

SCT, testis; dog. Tumor cells have both nuclear and cytoplasmic labeling, albeit cytoplasmic reactivity is weak. Inset: Detail of the immune reaction. Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

Fig. 6.

MGSCT, testis; dog. Sertoli cells are strongly labeled (nuclear), whereas germ cells are not. The center of this mass (asterisk) contains degenerated Sertoli cells positive for GATA-4 and germ cells negative for GATA-4. Inset: Detail of the immunolabeling. Immunoperoxidase-DAB for GATA-4. Hematoxylin counterstain.

In animals, GATA-4 expression was reported in adrenocortical neoplasms but not normal adrenocortical cells of ferrets; myocardial cells of dogs; Sertoli cells but not germ cells of pigs, cattle, and mice; Sertoli cells and Leydig (interstitial) cells of dogs, humans and mice. ^{3,4,6,7,10,13,14,16} In prepuberal pigs, GATA-4 is also weakly expressed in Leydig (interstitial) cells. ¹⁰ However, some human germ cell tumors (e.g., yolk sac carcinoma, ovarian dysgerminomas) express GATA-4. ^9,15 Our results are similar to those in humans in whom sex cord–stromal tumors express GATA-4 and GCTs do not. ⁶ Human ovarian sex cord–stromal cells (granulosa and theca) and surface epithelial ovarian cells and their tumors also express GATA-4. ^1,8,11 High expression of GATA-4 in ovarian sex cord tumors is considered a marker of poor prognosis. ¹ The intensity of staining varied in our tumor series. The significance of this is unknown and probably unrelated to tumor biologic behavior. Sex cord–stromal tumors of the canine testis are considered benign, with metastatic disease being uncommon or rare. ⁵ Evidence of metastatic disease was not apparent in the cases in this study. It is noteworthy that GATA-4 immunostaining facilitated the detection of MGSCTs when compared with hematoxylin and eosin stain because of the striking difference of staining between Sertoli cells and germ cells.

In summary, our results are similar to those in human testicular tumors in which Sertoli and LCTs, but not GCTs, consistently express GATA-4. However, there were some differences, such as cytoplasmic staining of some canine SCTs and many LCTs, and the expression of GATA-4 in normal germ cells of adult dogs. GATA-4 has been detected in human fetal and prepuberal testicular germ cells (spermatogonia) but not in adult testicles. ⁶ Nevertheless, some human germ cell tumors (e.g., yolk sac carcinoma, ovarian dysgerminomas) express GATA-4. ^9,15 Testicular GATA-4 expression among animal species varies and is most commonly detected in Sertoli cells (pigs, mice, cattle, and dog), Leydig cells, and germ cells (dogs). Our results indicate that 1) GATA-4 expression in the normal canine testis differs from that in testicular neoplasms in which germ cell tumors do not express this marker, 2) GATA-4 is a specific marker for sex cord–stromal tumors of the canine testis and can be used in conjunction with other markers in the characterization of testicular neoplasia, and 3) GATA-4 immunohistochemistry facilitates the distinction of MGSCTs from other testicular tumors.