Transcription Factor Adrenal 4 Binding Protein as a Marker of Adrenocortical Malignancy
HIRONOBU SASANO, MD, SOUICHIROU SHIZAWA, MD, TAKASHI SUZUKI, MD, KAZUTO TAKAYAMA, MD,
TAKAO FUKAYA, MD, KENICHIROU MOROHASHI, PHD, AND HIROSHI NAGURA, MD
Adrenal 4 binding protein (Ad4BP) is a transcription factor that regulates the expression of the steroidogenic enzymes and is ex- pressed primarily in steroidogenic cells. We immunolocalized Ad4BP in adrenocortical carcinoma (eight cases) and various malignancies that histologically simulate an adrenocortical carcinoma to evaluate the value of Ad4BP as an immunohistochemical marker of adrenocor- tical carcinoma. These malignancies examined were renal cell carci- noma (20 cases), hepatocellular carcinoma (10 cases), malignant mel- anoma (eight cases), ovarian (six cases) and uterine (three cases) clear cell carcinoma, large cell carcinoma of the lung (five cases), and pheochromocytoma (three cases). Nuclear Ad4BP immunoreac-
The nuclear protein adrenal 4 binding protein (Ad4BP) or steroid factor-1 is a transcription factor and controls the expression of all steroidogenic cytochrome P450 gene.1,2 Ad4BP regulates the gene expression through binding to the Ad4 site located at 330 base
noted the presence of Ad4BP messenger ribonucleic acid (mRNA) in the steroidogenic tissues including the testis, ovary, and adrenal glands.3 Ad4BP subsequently became exclusively localized in the steroidogenic cells except for gonadotropin-producing cells in the pitu- itary glands.5,6 In addition, Luo et al7 recently produced mice homozygously deficient in Ad4BP by using tar- geted gene disruption. These mice survived normally in utero, but all died by postnatal on day 8 from adreno- cortical insufficiency because adrenal glands were not developed in these animals.7 Based on these findings, Ad4BP is considered to play important roles in the em- bryonic differentiation and biological function of the primary steroidogenic tissues including adrenal cortex.
Adrenocortical carcinoma develops from adreno- cortical tissue, and exclusive localization of Ad4BP was reported in adrenocortical parenchymal cells of all the zonae.5 It is well known that biological features present in nonneoplastic adrenocortical cells including expres- sion of steroidogenic enzymes are altered through ma- lignant transformation.8,9 This makes it difficult to ob- tain specific adrenocortical tumor marker. In addition,
tivity was observed only in adrenocortical carcinoma cases but not in other tumors examined. Almost all of the adrenocortical carcinoma cells were immunohistochemically positive for Ad4BP including cells associated with bizarre nuclei. These results show that application of Ad4BP immunostain can contribute greatly to the differential diagno- sis of adrenocortical carcinoma. HUM PATHOL 26:1154-1156. Copy-
right @ 1995 by W.B. Saunders Company Key words: adrenal cortex, transcription factor, adrenal 4 binding protein, immunohistochemistry, diagnosis. Abbreviations: Ad4BP, adrenal 4 binding protein.
histopathologic diagnosis of adrenocortical carcinoma can be difficult, especially in the patients who do not manifest any adrenocortical hormonal abnormali- ties.10,11 In these cases, the malignancies that may be associated with histopathologic differential diagnosis at both primary and metastatic sites are renal cell carci- noma, hepatocellular carcinoma, clear cell carcinoma of the ovary and uterus, malignant melanoma, and large cell carcinoma of the lung and pheochromocytoma. Therefore, in this study, we examined the expression of Ad4BP in human adrenocortical carcinoma and the preceding malignancies to study the possible values of immunohistochemistry of Ad4BP in differential diagno- sis of adrenocortical carcinoma.
MATERIALS AND METHODS
Materials
Eight cases of adrenocortical carcinoma were examined in this study. Adrenocortical carcinomas were histopathologi- cally diagnosed based on the criteria of Weiss.11 Among carci- noma cases, five were associated with Cushing’s syndrome, and no adrenocortical hormonal abnormalities were observed in three cases. We also studied the following tumors: 20 cases of renal cell carcinoma, 10 cases of hepatocellular carcinoma, six cases of ovarian clear cell carcinoma, five cases of large cell carcinoma of the lung, three cases of uterine clear cell carcinoma, and three cases of phechromocytoma. The speci- mens were fixed in 8% paraformaldehyde or 10% formalin, pH 7.4, for 18 to 36 hours and were embedded in paraf- fin wax.
Immunohistochemistry
The immunohistochemical procedures were performed on 2.5-um-thick sections mounted on poly-L-lysine coated slides using the biotin-streptavidin amplified technique with a Histofine immunostaining kit (Nichirei, Tokyo, Japan). The staining procedure was performed as follows: (1) routine de-
From the Departments of Pathology, and Obstetrics and Gynecol- ogy, Tohoku University School of Medicine, Sendai, Japan; and the Department of Molecular Biology, Graduate School of Medical Sci- ence, Kyushu University, Higashi-ku, Fukuoka, Japan. Accepted for publication March 21, 1995.
Address correspondence and reprint requests to Hironobu Sa- sano, MD, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Japan 980.
Copyright @ 1995 by W.B. Saunders Company 0046-8177/95/2610-0004$5.00/0
paraffinization; (2) inactivation of endogenous peroxidase ac- tivity with 0.3% H2O2 in methyl alcohol for 30 minutes at 23°℃; (3) blocking with 1% goat serum for 45 minutes at 23℃; (4) incubation with the primary antibody at 4℃ for 18 hours; (5) incubation with biotinylated goat antirabbit anti- body for 30 minutes at 23°℃; (6) incubation with peroxidase- conjugated streptavidin for 30 minutes at 23℃; (7) colorimet- ric reaction with a solution containing 0.05% Tris-HCI, pH 7.6, 0.66 mol/L 3.3’-diaminobenzidine, and 2 mol/L H2O2; and (8) counterstaining with 1% methylgreen. The purifica- tion of Ad4BP and the subsequent production of a rabbit polyclonal antibody against Ad4BP and its characteristics have been described in detail by Morohashi et al.5 For positive control of immunostaining, rat ovary and adrenal were used. For negative control, the antibody preincubated with an ex- cessive amount of purified Ad4BP for 18 hours at 4℃, or 0.01M phosphate-buffered saline was used instead of primary antibody.
RESULTS
Immunoreactivity for Ad4BP was observed only in the cases of adrenocortical carcinoma (Figs 1 and 2). In these cases, immunoreactivity was localized exclu- sively in the nuclei of tumor cells (Figs 1 and 2). In adrenocortical carcinoma, intratumoral heterogeneity of relative Ad4BP immunointensity was observed, but almost all of the carcinoma cells in all eight cases showed nuclear immunoreactivity of Ad4BP. Ad4BP im- munoreactivity was observed in carcinoma cells with relatively low-grade nuclear atypia (Fig 1) but also in those with bizarre nuclei (Fig 2). The relative Ad4BP immunointensity was not different among areas with distinct architectures in the carcinoma, and between carcinomas associated with Cushing’s syndrome and no adrenocortical hormonal abnormalities. Ad4BP immu- noreactivity was not observed in any of the cases of malignancies examined in this study including the cases of renal cell carcinoma and hepatocellular carcinoma. Immunoreactivity observed previously was not detected in the negative control sections.
N
F
DISCUSSION
Adrenocortical carcinoma is a rare but an im- portant neoplasm for differential diagnosis when evalu- ating the patients with bulky tumors in the upper abdo- men or the patients clinically present with widespread metastasis of unknown primary lesion. Approximately 10% of the patients with adrenocortical carcinoma are not associated with clinical hormonal abnormalities, and clinical and histopathologic diagnosis can be diffi- cult in these cases.12 Diagnosis of adrenocortical carci- noma is important in these patients because op’-DDD treatment results in a small but significant increase in mean survival times in the patients with adrenocortical carcinoma.13
The two most important primary neoplasms in the differential diagnosis of adrenocortical carcinoma are renal cell and hepatocellular carcinoma.9 In addition, the differential diagnosis between adrenocortical carci- noma and metastatic tumors, such as malignant mela- noma or large cell carcinoma of the lung or clear cell carcinoma of the ovary or uterus and phechromocy- toma, can be a difficult one on occasion.9 It is true that careful histological examination and detection of biological features, such as «-1 antitrypsin or «-fetopro- tein immunoreactivity in hepatocellular carcinoma, abundant glycogen in the clear cell types of renal cell carcinoma and clear cell carcinoma of the ovary and uterus, and S-100 and HMB-45 immunoreactivity in ma- lignant melanoma and chromagranin in phechromocy- toma, can resolve the diagnostic dilemma between adre- nocortical carcinoma and other tumors in most cases. However, it is also true that specific adrenocortical tu- mor marker can contribute greatly to the differential diagnosis described earlier.
The presence of abundant well-developed smooth endoplasmic reticulum and mitochondria with tubular or tubulovesicular cristae by electron microscopic ob- servation strongly indicates adrenocortical carcinoma. However, these findings are observed only in one half
of the cases of adrenocortical carcinoma.9,14 Demonstra- tion of steroidogenic enzymes, especially the enzymes present in only the adrenal cortex, such as P450cl1 (118-hydroxylase),15 can be of great help in the diagno- sis of adrenocortical carcinoma. However, the patterns of expression of steroidogenic enzymes in adrenocorti- cal carcinoma are different from adrenocortical ade- noma, and several carcinoma cells do not express steroidogenic enzymes involved in corticosteroidogen- esis.8 Recently, Tartour et al16 reported that nuclear immunoreactivity recognized by the D11 monoclonal antibody was highly specific for adrenocortical carci- noma, but immunoreactivity was only observed in 44% of the carcinoma and was restricted to well-differenti- ated types of adrenocortical carcinoma.
Our present study of Ad4BP immunohistochemis- try showed that only adrenocortical carcinomas were positive for Ad4BP among the tumors studied, and al- most all adrenocortical carcinoma cells showed nuclear Ad4BP immunoreactivity. Immunoreactivity of Ad4BP was also reported in Leydig cells of the testis,5 theca and granulosa cells of the ovary,5 and gonadotropin- producing cells of the pituitary.6 In addition, we re- cently showed nuclear Ad4BP immunoreactivity in ste- roid-producing cells of ovarian sex cord-stromal tumor including steroid cell tumor and not otherwise specified tumors of the ovary that can metastasize to the upper abdomen.17 Therefore, Ad4BP may not be an exclusive marker of adrenocortical carcinoma; however, from practical standpoints, immunohistochemical evaluation of Ad4BP is considered to contribute greatly to the definitive diagnosis of adrenocortical carcinoma among various human malignancies.
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