Immunohistochemical Expression of Epidermal Growth Factor Receptors in Human Adrenocortical Carcinoma
TAKIHIRO KAMIO, MD, KAZUTO SHIGEMATSU, MD, HOUHEI SOU, MD, KIOKO KAWAI, MD, AND HIDEO TSUCHIYAMA, MD
Epidermal growth factor (EGF) receptors were examined immu- nohistochemically in 64 adrenocortical carcinomas obtained at autopsy, and in 23 adrenocortical adenomas and seven pheochro- mocytomas obtained during surgery. In the nonneoplastic adre- nal gland, EGF receptors were scattered to the zona glomerulosa, zona fasciculata, and zona reticularis. Adrenocortical carcinomas (63 of 64), more than adrenocortical adenomas (10 of 23) or pheochromocytomas (four of seven), stained positively for EGF receptors (P < . 01). The immunoreactivity was limited to the cytoplasm, cell membrane, and chromatin. When the antibody was immunoabsorbed with an excess of immunogen peptide, there was no evidence of immunostaining. The adrenocortical carcinomas could be classified into 16 cases of the well- differentiated type, 33 cases of the moderately differentiated type, and 15 cases of the poorly differentiated type. There was no relationship between histologic grading and staining intensity of the EGF receptors. On the other hand, more than 80% of the cases of adrenocortical carcinomas revealed a moderate to high inten- sity for EGF receptors. In 62 of the 64 patients, there was already metastases to other organs. We conclude that the expression of EGF receptors is associated with tumor growth and/or metastatic potential in adrenocortical carcinoma. HUM PATHOL 21:277- 282. @ 1990 by W.B. Saunders Company.
Epidermal growth factor (EGF) isolated from mouse submandibular gland1,2 and human urine3 is known to mediate cell proliferation4,5 by interacting with specific receptors.5-7 Receptors for EGF (EGFr) on the cell surface membrane6.7 are transmembrane glycoproteins of about 170 kilodalton,8 and the bind- ing of EGF to its cell surface receptors triggers induc- tion of an intracellular tyrosin kinase activity,9 which induces cell division.10 The EGF-EGFr complexes formed after binding are internalized in the cytosol, and have been termed receptor endocytosis.6,11,12 The tyrosine kinase oncogene family, v-erbB onco- gene, resembles part of the gene encoding the inter- nal domain and transmembrane portion of EGFr, 13-15 thereby suggesting that EGF may contrib- ute to the tumorigenesis. A high level of EGFr is ex- pressed in human squamous cell carcinomas of the lung, 14,16 breast cancers,17 and brain tumors. 18,19
From the Department of Pathology 2, Nagasaki University School of Medicine, Nagasaki, Japan. Accepted for publication July 21, 1989.
Key words: epidermal growth factor receptor, human, adrenal gland, carcinoma.
Address correspondence and reprint requests to Kazuto Shigematsu, MD, Department of Pathology 2, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan. @ 1990 by W.B. Saunders Company. 0046-8177/90/2103-0004$5.00/0
Gene amplification of EGFr has also been noted in several tumors,14,18 and alterations in chromosome 7 may be involved.20
In the adrenal gland, EGF acts as a steroido- genic21 and mitogenic22 factor, and the existence of EGFr in the rat adrenal gland has been demon- strated by using an in vivo autoradiographic technique.23 However, little is known of the activity of EGFr in primary adrenocortical carcinomas, a rare neoplasm. We used an immunohistochemical method to determine whether EGFr are expressed in human adrenocortical carcinoma, and our findings were compared with those obtained in other cases of adre- nal tumors, including adrenocortical adenoma and pheochromocytoma.
MATERIALS AND METHODS
Tissue Samples
Of the paraffin-embedded human adrenocortical car- cinomas listed in the annual report of Pathological Autopsy Cases in Japan between 1965 and 1982, 98 were collected. Among them, 64 cases confirmed as adrenocortical carci- noma by hematoxylin-eosin stain, periodic acid-Schiff reac- tion, and silver impregnation were investigated. There was no overt autolysis. The adrenocortical carcinomas were then graded into well-differentiated type (GI), moderately differentiated type (G2), and poorly differentiated type (G3), on the basis of how closely the cells resembled normal cortical cells.24 Twenty-three cases of adrenocortical adeno- mas (nine cases of Cushing’s syndrome, 11 cases of Conn’s syndrome, and three cases of nonfunctioning) and seven cases of pheochromocytomas were examined at Nagasaki University School of Medicine. Normal adrenal tissue ad- hering to the tumor served as the normal control.
Immunohistochemical Staining of Tissues
Formalin-fixed, paraffin-embedded tissues were cut on a microtome to a thickness of 5 um and thaw-mounted onto gelatin-coated glass slides. Deparaffinized sections were in- cubated in 0.3% H2O2 solution for 30 minutes at room temperature to abolish endogenous peroxidase activity. The avidin-biotin complex method was then used (Vecta- stain avidin-biotin complex kit, Vector Laboratories, Inc, Burlingame, CA). After treatment with 20% normal goat serum for 60 minutes, the tissue sections were allowed to react for 60 minutes at room temperature with anti-human EGFr antibody (Cambridge Research Biochemicals, Inc, Valley Stream, NY; diluted 1:49, 1:99 and 1:199 with 0.01 mol/L phosphate buffered saline, pH 7.6). Subsequently, the tissue sections were incubated in biotinylated goat anti- rabbit immunoglobulin at room temperature for 30 min- utes. A complex of avidin-biotin-horseradish peroxidase
was applied, and the preparation was incubated for 30 min- utes. The antibody-bound peroxidase was revealed with 0.04% diaminobenzidine (Sigma Chemical Co, St Louis, MO). The tissue sections were then counterstained with he- matoxylin and dehydrated in a graded series of ethanol.
Immunospecificity Studies
To determine specific binding, non-immune rabbit sera were substituted for primary antibody, and immuno- staining was prevented by preincubation of primary anti- body with an excess of immunogen peptide (Cambridge Research Biochemicals, Inc).
Evaluation of Staining Intensity
Adrenocortical carcinomas which stained positively for EGFr were semiquantitatively graded on a rating scale ranging from 1 (mild, 1% to 10% positive cells) to 2 (mod- erate, 10% to 50% positive cells), to 3 (high, 50% to 100% positive cells). In cases with no reaction, a score of 0 was recorded.
Statistical Analysis
The data were assessed by x2 test and Kendall’s rank ☒ correlation test (Kendall tau).
RESULTS
Sixteen of the 64 patients had functioning adre- nocortical carcinomas: 13 with Cushing’s syndrome, one with Conn’s syndrome, and two with adrenogen- ital syndrome (Table 1). Sixty-two had metastases to other organs, and 78.3% of the patients with adreno- cortical carcinomas died within I year of the onset of symptoms.
In the nonneoplastic adrenal gland, EGFr were faintly expressed on the zona glomerulosa cells (Fig 1, top), as well as on the cells of the zona reticularis (Fig 1, bottom). Stained cells were scattered among zona fascicular tissues. Immunostaining in the adrenal me- dulla was nil. As shown in Table 1, EGFr immunore- activity was present in 63 of 64 adrenocortical carci- nomas (98.4%), and only in one did the primary site
| Histologic Type | No. of Cases | EGFr Positivity (%) |
|---|---|---|
| Adrenocortical carcinoma | 64 | 63 (98.4) |
| Cushing's syndrome | 13 | 13 |
| Conn's syndrome | 1 | 1 |
| Adrenogenital syndrome | 2 | 2 |
| Nonfunctioning | 48 | 47 |
| Adrenocortical adenoma | 23 | 10 (43.5)* |
| Cushing's syndrome | 9 | 3 |
| Conn's syndrome | 11 | 5 |
| Nonfunctioning | 3 | 2 |
| Pheochromocytoma | 7 | 4 (57.1)+ |
* x2 = 33.9 (carcinomas v adenomas), P < . 01. ☒
t x2 = 13.2 (carcinomas v pheochromocytomas), P < . 01. ☒
show no reaction for EGFr. The frequency of EGFr expression in the adrenocortical carcinomas was sig- nificantly higher than in the adrenocortical adenomas (10 of 23, 43.5%) and pheochromocytomas (four of seven, 57.1%; P < . 01). In adrenocortical adenomas, some compact-type cells, but not clear-type cells, gave a positive reaction. Immunoreactivity in the pheochromocytomas was in the form of diffuse- positive cells. The frequency of EGFr expression be- tween adrenocortical adenomas and pheochromocy- tomas was not statistically significant. The majority of tumor cells positive for EGFr exhibited heterogeneity in a fine granular pattern in the cytoplasm (Fig 2, top left). In some areas, the immunoreaction was limited to the cell membrane (Fig 2, top right) and to the chromatin (Fig 2, bottom left). Blood vessels and ne- crotic areas were negative for EGFr. When the pri- mary antibody was preincubated with an excess of immunogen peptide, there was no evidence of posi- tive staining (Fig 2, bottom right).
The adrenocortical carcinoma tissues were then subjected to histologic grading according to cell dif- ferentiation (Table 2). Sixteen of 64 tissues were clas- sified to the GI group, 33 to the G2 group, and 15 to the G3 group. Thirty-eight of the carcinoma tissues taken from the primary site (79.2%) and all 16 of those taken from metastatic sites (100%) had a mod- erate to high intensity of EGFr. There was no signif- icant relationship between intensity of the EGFr stain- ing (Kendall’s tau = 0.185; P = . 2468). In addition, there was no difference in intensity of the EGFr stain- ing between nonfunctioning and functioning adreno- cortical carcinomas (Kendall’s tau = 0.204; P = .4030, Table 3).
DISCUSSION
We obtained immunohistochemical evidence that human adrenal tumors, including adrenocortical car- cinomas, adrenocortical adenomas, and pheochromo- cytomas, contain EGFr-immunoreactive materials. To differentiate the tissue preparations,25-28 only forma- lin-fixed and paraffin-embedded tissues were used. In the initial experiment, staining was prevented by preincubation of the antibody with an excess of im- munogen peptide, and as immunoreactivity was un- detectable in blood vessels, the reactions we later ob- tained were considered to exhibit specific binding.
The cytomorphologic findings showed immuno- reactivity mostly in the cytoplasm and cell membrane. This phenomenon could be due to internalization of EGF-EGFr complexes into the cytoplasm via recep- tor-mediated endocytosis.6,11,12 In addition, the EGFr antibody used in this study bound to the nuclear chromatin. Although receptor proteins taken up into the cytoplasm are, in general, degraded in lysosomes and recycled and reintegrated in the plasmalemma,29 Rakowicz-Szulczynska et al30 found that EGF was tightly bound to chromatin, and that this binding of EGF was prevented by monoclonal antibody against
EGFr, thereby indicating the presence of EGFr-like molecules as well as nerve growth factor receptors in the chromatin.
Faint expression of EGFr was also observed in the nonneoplastic adrenal cortex. However, the con- stitutive expression of growth factor receptors results in malignant transformation and cell growth.29 Of the adrenocortical carcinomas investigated, 98.4% re- vealed a strong positivity for EGFr expression, and the frequency was significantly higher than that seen in benign adrenal tumors, such as adrenocortical ad- enomas and pheochromocytomas. Taken in conjunc- tion with the evidence that overexpression of EGFr and amplification of the EGFr gene occur in some human tumors, including carcinomas,14,16-19 the pos-
sibility that EGFr-mediated pathophysiologic pro- cesses are associated with growth of adrenocortical carcinomas would have to be considered.
With regard to assessments of histologic grading, we found no correlation with EGFr expression, in contrast to reported results.17,25,27 Other investiga- tors have reported that EGFr is associated with the advanced stage of tumors and with metastatic potential.26,28 Still others have found no relationship between EGFr and tumor stage or activity.31 We did not investigate whether EGFr could serve as a prog- nostic parameter in adrenocortical carcinomas; how- ever, our observation that over 80% of the cases we examined showed a moderate to high intensity of EGFr staining, with no correlation to histologic grad-
ing, may be because metastases to other organs had already occurred in 62 of 64 of the patients.
Compact-type cells, but not clear-type cells, in ad- renocortical adenomas revealed the EGFr immuno- reactivity. The clear-type cells contain an amount of
lipid droplets and scanty organellae, whereas the compact-type cells show a marked development of organellae.32.33 Therefore, the expression of EGFr in the clear-type cells may be difficult to detect using an immunohistochemical technique. On the other hand,
| Staining Intensity | |||||
|---|---|---|---|---|---|
| 0 (%) | 1 (%) | 2 (%) | 3 (%) | Total | |
| Primary site | |||||
| Grade | |||||
| G1 | 0 (0.0) | 5 (35.7) | 4 (28.6) | 5 (35.7) | 14 |
| G2 | 1 (4.8) | 4 (19.0) | 4 (19.0) | 12 (57.1) | 21 |
| G3 | 0 (0.0) | 0 (0.0) | 6 (46.2) | 7 (53.8) | 13 |
| Subtotal | 1 (2.1) | 9 (18.8) | 14 (29.2) | 24 (50.0) | 48 |
| Metastatic site | |||||
| Grade | |||||
| G1 | 0 (0.0) | 0 (0.0) | 0 (0.0) | 2 (100) | 2 |
| G2 | 0 (0.0) | 0 (0.0) | 5 (41.7) | 7 (58.3) | 12 |
| G3 | 0 (0.0) | 0 (0.0) | 1 (50.0) | 1 (50.0) | 2 |
| Subtotal | 0 (0.0) | 0 (0.0) | 6 (37.5) | 10 (62.5) | 16 |
| Total | 1 (1.6) | 9 (14.1) | 20 (31.2) | 34 (53.1) | 64 |
NOTE. Kendall’s tau: . 185, P = . 2468.
EGF RECEPTORS IN ADRENOCORTICAL CARCINOMA (Kamio et al)
| Staining Intensity | |||||
|---|---|---|---|---|---|
| 0 (%) | 1 (%) | 2 (%) | 3 (%) | Total | |
| Nonfunctioning | 1 (2.1) | 7 (14.6) | 16 (33.3) | 24 (50.0) | 48 |
| Functioning | 0 (0.0) | 2 (12.5) | 4 (25.0) | 10 (62.5) | 16 |
NOTE. Kendall’s tau: . 204, P = . 4030.
the possibility that the presence of EGFr may be as- sociated with active steroidogenesis in the compact- type cells21,33 could be considered.
Some pheochromocytoma cells gave a positive re- action, while immunostaining was not observed in the nonneoplastic adrenal medulla. It has been demon- strated that EGF will bind to rat pheochromocytoma (PC12) cells.34 These observations support the view that EGF induces differentiation of pheochromocy- toma cells, and increases the secretion of catechola- mines from pheochromocytoma cells.35
In conclusion, adrenocortical carcinomas express EGFr at a high frequency. Expression of EGFr was independent of the histologic grading, but may be associated with tumor growth or metastatic potential. Malignant cells may secrete a hormone-like sub- stance, which functions on cell surface receptors, by an autocrine mechanism.36 In addition, many types of malignant tumors produce transforming growth factor-alpha to bind to EGFr.37 Whether EGF or transforming growth factor-alpha is secreted by ad- renocortical carcinoma cells is the subject of ongoing study.
Acknowledgment. We thank M. Ohara for critical com- ments and Y. Yamashita and S. Nakanose for technical as- sistance.
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