ESTROGEN-PRODUCING ADRENOCORTICAL CARCINOMA A Light and Electron Microscopic Study
Kouki INAI, Kazuhiko OHE, Shoji TOKUOKA, and Yukitaka MIYACHI*
Second Department of Pathology, Hiroshima University School of Medicine, Hiroshima
* Department of Nuclear Medicine, Shizuoka Prefectural Hospital, Shizuoka
A case of adrenocortical carcinoma with feminization seen in a man aged 35, is reported. The levels of estrone (E1) and estradiol (E2) in the venous blood draining the tumor were high, and it was confirmed by in vitro assay of tumor cells taken from the primary tumor of the left adrenal gland that the tumor produced estrone. The light microscopic examination demonstrated that the primary tumor was composed of mixture of large cells with pleomorphic nuclei and vacuolated cytoplasm and uniform cells with ovoid nuclei and eosinophilic cytoplasm. The electron microscopic examination on the latter cells revealed numerous large and irregularly shaped mitochondria with mostly tubular or lamellar and occasionally vesicular cristae and electron-dense matrix, well- developed smooth-surfaced endoplasmic reticulum in the cytoplasm. However, lipid droplets and lysosomes or lipofuscin granules were scanty. From these findings, it is suggested that cells of the present tumor have characteristics of those in the zona reticularis of the adrenal cortex as well as in the fetal cortex, and the functional property of this tumor is well correlated with its morphological features. ACTA PATHOL. JPN. 34: 693~704, 1984.
Introduction
Clinical hormonal symptoms as functional phenomena of adrenocortical carci- noma are noted in 60% of 90 cases in Japanese recent reports.17 The most frequent symptom is Cushing’s syndrome, followed by virilization and mixed form of Cushing’s syndrome and virilization, and feminization is very rare.
Because of the difficulty of recognizing feminizing symptoms in postpuberal women, the reports of adrenocortical carcinoma with feminization (ACCF) are limited to postpuberal men and female children. GABRILOVE6 in 1965 primarily reviewed 52 cases of feminizing adrenocortical tumors in the male, including 41 cases of ACCF. Additional nine cases of ACCF were added by the same author in 1970.7 In Japan,
only two cases of ACCF have been reported according to SHIMAZAKI et al.17
From the view point of the morphological study on ACCF, the majority of these reports were based on the clinical and histological appearances of the tumor and only one reported by MITSCHKE et al.13 has so far dealt with not only the histological appearance of the tumor but also the ultrastructural morphology of the tumor cells.
The purpose of this report is to present histological as well as ultrastructural findings of the primary tumor in a case of ACCF and to make a comment on the significance of characteristic ultrastructural features.
Case Report
A Japanese man, 35 years of age, complained in May of 1976 of left gynecomastia and reduced libido. It appeared that the administration of Mastizol, an synthesized androgenic hormone, in the amount of 100 mg per week for successive five months was successful and his left gynecomastia disappeared. About two years later, he began to realize a general tiredness, weight loss, and tenderness in the left upper quadrant of the abdomen, and was admitted to the hospital. Physical examinations at that time revealed bilateral gynecomastia and a palpable elastic soft mass in the left upper quadrant of the abdomen. The blood pressure was 120/80 mmHg.
Laboratory examinations of the peripheral blood revealed high levels of estrone (E,) as well as estradiol (E2), as shown in Table 1, and low levels of FSH, LH, and
| Pre-ope. | Post-ope. | |
|---|---|---|
| Cortisol (2g/dl) | 14.2 | 13.0 |
| Testosterone (ng/d)) | 176.0 | 675.5 |
| Dihydrotestosterone (ng/dl) | N.D. | 36.8 |
| Androstenedione (ng/dl) | 781.8 | 60.8 |
| Aldosterone (pg/ml) | 34.6 | 41.6 |
| Progesterone (ng/ml) | 0.49 | 0.64 |
| E, (pg/ml) | 1545 | <5 |
| E2 (pg/ml) | 300 | <5 |
| E, (pg/ml) | 5 | <5 |
| LH (ng/ml) | 10.0 | 68.0 |
| FSH (ng/ml) | 17.0 | 166.0 |
| Prolactin (ng/ml) | 20.0 | 21.0 |
| TSH («U/ml) | 8.0 | 5.0 |
| GH (ng/ml) | 1.0 | 4.0 |
| 170 HCS (mg/24 hrs.) | 26.0 | 4.4 |
| 17 KS (mg/24 hrs.) | 27.0 | 3.7 |
N.D. = Not Done
| E, (pg/ml) | E2 (pg/ml) | E3 (pg/ml) | Cortisol (µg/ml) | |
|---|---|---|---|---|
| Heart | 831 | 81 | 5 | 5.8 |
| Vena cava inf. | 708 | 102 | 5 | 7.6 |
| Lt. Renal vein | 2612 | 200 | 5 | 9.8 |
| Lt. adrenal vein | 1397 | 301 | 5 | 14.8 |
testosterone. The levels of cortisol and aldosterone appeared to be within the normal limits. Urinary 17-OHCS excretion was increased, suggesting the reduced activity of 116-hydroxylase in the synthetic pathway.
An abdominal exploration revealed a large tumor noted in the left suprarenal region, involving the left adrenal gland. A total resection of the tumor with the adrenal gland was performed. The levels of estrogens and cortisol in the venous blood draining the tumor at the time of surgery are shown in Table 2.
In the meantime after the removal of the tumor, bilateral gynecomastia dis- appeared, and libido and potency improved. Administration of o,p-DDD, an inhibi- tor of adrenocorticosteroid hormone biosynthesis, was begun. About one year later, however, the patient noted pain in the back and chest. A recurrent tumor in the same area of the retroperitoneal space was detected by an angiography, and distant metastases to the lungs and the cerebrum were shown in chest X-ray films and a CAT scan. He expired in July of 1979, 15 months after the surgical removal of the primary tumor, and was autopsied.
At the time of autopsy, the recurred tumor was present in the suprarenal region, measured 12x9x8 cm in size and weighed 480 g. The metastases were found in the bilateral lungs and the right cerebral hemisphere. Prominent bilateral gynecomastia and hypospermatogenesis of the testes were also noted.
Materials and Methods
Tissue sections from the tumor removed surgically was prepared as usual for the light microscopic examination. Besides, tissues taken from some parts of the tumor were fixed in 2% glutaraldehyde, postfixed in 1% osmium tetraoxide, and embedded in Epon 812. Ultra-thin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi HU7 electron microscope.
Moreover, a piece of the tumor was submitted for in vitro study. As a primary culture of tumor cells, minced tumor tissue was dispersed in Ham F medium containg 2.5% fetal calf serum and incubated at 37℃ in a humidified atmosphere of 5% CO2 concentration for 4 weeks. The media used were changed every 3 or 4 days and submitted for the measurement of estrogen by means of radioimmunoassay. Cultured cells were also prepared for electron microscopic examination.
Estrogen radioimmunoassay was performed as follows.11 Serum samples (0.5 ml) and incubation media (1 ml) were extracted twice with ten volumes of ethyl ether. The ethyl ether was evaporated to dryness under nitrogen in a 37℃ water bath. The extracts, dissolved in 0.2 ml of benzene : methanol (9 : 1), were applied on a Sephadex LH-20 (Pharmacia Fine Chemicals, New Jersey) column and eluated with the same solvent.3 The eluate was evaporated to dryness and redissolved in 2 ml of ethanol, appropriate portions of which were radioimmunoassayed for E1, E2 and E3 as described previously.14 Coefficiency of variation for inter- and intra-assay variations were both 5-15%.
Pathology
Gross findings : The primary tumor removed surgically measured 14x 11 x 7 cm in size and weighed 800 g. The tumor was relatively well defined with thin capsule. On cutting, the tumor was soft, tan to brown in color and solid. There were multiple areas of massive necrosis and hemorrhage particularly in the central region (Fig. 1.).
0
1 2 3 4 5 6 7 8 9 10 11 12 13
14
1
Light microscopy : The tumor showed two different histological appearances in different areas. In the subcapsular areas, tumor cells were large and polygonal and showed pleomorphic nuclei and abundant vacuolated cytoplasm and formed columnar or fasciculated patterns that were separated by fine stroma. Mitotic figures were very few (Figs. 2a, b). On the other hand , in the inner area, the tumor tissue consisted of relatively uniform cells with ovoid nuclei and eosinophilic cytoplasm, forming solid nests with sinusoidal vessels in scanty stroma. In areas of stromal myxoid change, a pseudotubular arrangement of tumor cells was seen. Mitotic figures were frequently seen in these cells (Figs. 3a, b).
Within the capsule, findings of vascular invasion of pleomorphic tumor cells were seen. Histological diagnosis of adrenocortical carcinoma was made. A small part of the left adrenal gland, found within the capsule, was atrophic in each of three layers.
Fig. 2. (a) Tumor cells forming columnar or fasciculated pattern in the subcapsular area of the tumor. H.E., × 50. (b) The tumor cells are large and polygonal with pleomorphic nuclei and abundant vacuolar cytoplasm. H.E., × 200.
Fig. 3. (a) Tumor cells forming solid nests with sinusoidal vessels in the inner area of the tumor. (b) The tumor cells are relatively uniform with ovoid nuclei and eosinophilic cytoplasm. Many mitotic figures are noted. H.E., × 200.
2.
3.
2
26
3b
4
5
Fig. 4. Nests of the tumor cells consist of clear cells and dark cells. The cytoplasm of one cell protrudes into that of its neighbor with occasional desmosomes. x 7,020. Fig. 5. Most of the mitochondria are large and irregularly shaped and the cristae are mostly of tubular or lamellar type and occasionally of vesicular. x 27,300. Fig. 6. Smooth-surfaced endoplasmic reti- culum are sometimes arranged in a circular fashion and rough surfaced ones are often arranged in stack-like fashion. × 11,700.
6
7
50
100 200 ¿ Imedium
90
Q
o
80
9
O
70
60
Medium
5
8
Bound Percent
50
5
Estrone
40
30
20
10
100
1000
1000
Estrone (pg/tube)
Electron microscopy : Electron microscopical examinations were made only on the tumor cells that were uniform with eosinophilic cytoplasm and distributed in the inner area of the tumor as above described. Although nests of these cells appeared to consist of two kinds of cells, clear and dark, under the electron microscope, cytoplasmic details of both cells were not so different from each other.
The cytoplasm of one cell protruded into that of its neighbor with occasional desmosomes. In general, the tumor cells were polygonal and their nuclei were round or irregular with indentations. The nucleoli were small and inconspicuous. The
7(0)
9
10
chromatin was aggregated along the nuclear membrane (Fig. 4).
Intracytoplasmic organelles were abundant. Mitochondria were large and irre- gularly shaped and often clustered. The cristae were mostly of tubular or lamellar type and occasionally of vesicular. The matrix of the mitochondria was homogenous- ly electron-dense, with no granular or crystalloid structures (Fig. 5).
Profiles of well-developed smooth-surfaced endoplasmic reticulum, sometimes of circular arrangement were more abundant than rough-surfaced endoplasmic reticulum, often of stack-like arrangement (Fig. 6). The Golgi complex was very few.
Lipid droplets of moderate density were scant and distributed mainly in the peripheral areas of the cytoplasm. A few lysosomes or lipofuscin granules were found throughout the cytoplasm (Fig. 4).
The basement membrane of clustered tumor cells was often disrupted. Sinusoidal vessels in tumor stroma were lined by a fenestrated endothelium with continuous basement membrane. Spaces between clustered tumor cells and sinusoidal vessels appeared to be widened and occupied by mucoid materials (Fig.7).
Short-term culture of tumor cells : Each medium used for 4 weeks culture of tumor cells was submitted for a radioimmunoassay of estrogen. The standard curve of E, in the medium was parallel to the dilution curve of the medium, as shown in Figure 8, indicating that E, was produced by the tumor cells in culture. The concentration of E, in the medium was very high, 2 ng/ml.
An ultrastructural observation on cultured tumor cells for 4 weeks showed many cytoplasmic processes and abundant intracytoplasmic organelles that were similar to those seen in the primary tumor tissue. However, mitochondria and profiles of smooth-surfaced endoplasmic reticulum were more abundant than the primary tumor and profiles of rough-surfaced endoplasmic reticulum decreased. The cristae of mitochondria were tubular or lamellar. Lysosomes or lipofuscin granules were more abundant. These findings of cultured tumor cells were suggestive of preservation of estrogen-producing ability. In the nuclei, euchromatin was more prominent than the primary tumor, suggestive of nuclear activity (Figs. 9, 10).
Discussion
As previously mentioned, only a case report on an ultrastructural observation of ACCF has been available in the literature at present. In that report by MITSCHKE et al.,13 the tumor, weighing 120 g, was smaller than the present case. Histologically it was made up of uniform cells with vesicular nuclei and weakly eosinophilic cytoplasm, and mitotic figures were not demonstrable. Accordingly these tumor cells were different from the cells, observed ultrastructurally in the present case. However, it was confirmed by in vitro assay that the tumor cells of the present case produced estrone, and so it is worth to compare with the report by MITSCHKE et al.13
According to their report, there were a lot of mitochondria with various features in tumor cells. Predominant ones were large and round to oval with light appearing
matrix and lamellar cristae sparsely distributed. Most of them contained electron- dense granules, suggestive of degenerative process. The relationship between specific mitochondrial alterations and enzymatic abnormalities, responsible for accelerated estrogen production, has been of great interest, but this has not been documented in their report. In the present case, no obviously different types of mitochondria were detected. Moreover, the cristae were mostly of tubular or lamellar and occasionally of vesicular, and the matrix was highly electron-dense with no granules. These findings were rather different from those described by MITSCHKE et al. With respect to other organelles, MITSCHKE et al. noted that smooth-surfaced endoplasmic reticulum was poorly developed in the tumor cells containing a lot of mitochondria. On the con- trary, most of the tumor cells in the present case showed well-developed smooth- surfaced endoplasmic reticulum, occasionally arranged in a circular fashion, however, no relationship to mitochondrial number was noted.
Findings of few lipid droplets and lysosomes or lipofuscin granules were of characteristic and accord with the previous report.13 Scarcity of lipid droplets in the adrenal tumors has been regarded to be a characteristic indication for high cholesterol metabolism, as compared with normal adrenocortical cells.12 The findings in the present case might support this view.
In estrogen-producing tumors, it is supposed that conversion from androgens to estrogens occurs6 and, therefore, it may be no wonder that there is a morphological parallelism between virilizing and feminizing adrenocortical tumors. From this view point, ultrastructural findings of virilizing adrenocortical tumors as well as feminizing ones are worthy to compare with each other. Ultrastructural studies on virilizing adrenocortical tumors have been available.1,2,5,8,9,16,18 VALENTE et al.18 reported two cases of androgen-secreting adrenocortical carcinoma composed of tumor cells with histological and ultrastructural features resembling cells of the zona reticularis in the human adrenal cortex. The similarity was confirmed in the findings of numerous mitochondria with lamellar or tubular cristae, abundant smooth-surfaced endoplasmic reticulum, variable amount of lipofuscin granules and scanty lipid droplets. Also, in virilizing adenomas the amount of lipofuscin granules or lysosomes appears to be variable. AIBA et al.1 suggested that the scantiness of lipofuscin granules did not negate the possibility of the zona reticularis origin of adenoma cells according to ultrastructural as well as histochemical observations. On the basis of the above information, it is suggested that ultrastructural findings of the present case showed no apparent differences from those of virilizing adrenocortical tumors.
Another attention is called on mitochondria, large in size and/or irregular in shape, of the present case. Mitochondria in cells of the fetal cortex are varied in size and the cristae are of lamellar or tubular10. In addition, smooth-surfaced endoplasmic reticulum is well developed. It was thought, therefore, that the tumor cells of the present case also resemble the cells of the fetal cortex ultrastructurally. Moreover, on the light microscopic observations, the tumor cells in the inner area of the tumor were uniform with ovoid nuclei and eosinophilic cytoplasm, suggestive of resemblances to
34(3) : 1984
cells of the fetal cortex. As ultrastructural observation on the large tumor cells in the subcapsular area of the tumor was not performed unfortunately, a comparative study on therse two different cell types was unable to be made.
In the present study, the primary culture of tumor cells apparently demonstrated production of estrone. Ultrastructurally the tumor cells after 4 weeks culture showed similar cytoplasmic characteristics to the primary tumor. In general, cultured cells reveal dedifferentiated appearances, but in the present case, the cytoplasmic organelles were well preserved, suggestive of functional ability. In recent years, FANG4 esta- blished a new cell line, which originated from ACCF in a man of 30 years of age, and O’HARE et al.15 reported a study on cell culture of 9 cases of functional adrenocortical tumors. In these reports as well as the present case, careful procedures enabled the cultured tumor cells to retain the functional attributes, which were expressed in vivo. This culture method may add a further progress in endocrinological research on ACCF, such as a pathway of steroidgenesis or response to ACTH or other stimulants.
Acknowledgement : The authors thanks Dr. T. OKADA, Mrs. T. KONDO and Miss F. MATSUI for their assistance in procedure of cell culture.
References
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