Adrenocortical Carcinomas: An Ultrastructural Study of 22 Cases
Elvio G. Silva, MD, Bruce Mackay, MD, N. A. Samaan, MD, and R. C. Hickey, MD
Departments of Pathology, Medicine, and Surgery, The University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, Houston, Texas 77030
Twenty-eight adrenocortical carcinomas from 22 pa- tients have been studied by light and electron micros- copy. The objectives were to document the range of fine structure, to establish ultrastructural criteria for diagnosis, and to relate the morphology to the pres- ence or lack of functional activity. The distinctive ultrastructural features of steroid-forming cells, smooth endoplasmic reticulum and tubular mitochon- drial cristae, were only present in some cells of some of the cases. Sequential specimens (up to four) from a single patient demonstrated changes in the tumor cell morphology with progression, including marked accu- mulation of cytoplasmic glycogen.
KEY WORDS: adrenocortical carcinoma, ultrastruc- ture, cytoplasmic glycogen, matrical granules.
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INTRODUCTION
Carcinomas of the adrenal cortex are relatively uncommon, and there is limited published in- formation on their ultrastructure.1-4 In this study of 22 cases, the basic objectives were to document the range of fine structure presented by the tumor cells, relate the ultrastructural findings to the light microscopic histology, and determine the specificity of the fine structural features and their value in differential diagnosis. Sequential specimens, including four accessions over a period of years from one patient, pro- vided the opportunity to identify changes in morphology that accompanied progression of the tumor. The medical records of the patients were reviewed and an attempt was made to relate the structure of a tumor with presence or lack of functional activity.
MATERIAL AND METHODS
Twenty-eight specimens of adrenocortical car- cinomas from 22 patients were studied by light and electron microscopy. The accessions in-
cluded 15 primary and 13 metastatic tumors; 5 patients had received radiotherapy or chemo- therapy, or both. Multiple specimens were ob- tained from 3 patients, including four from one, three from another, and two from a third. In 5 patients, there was evidence of hormonal activity (3 Cushing’s, 1 virilism, and 1 Cushing’s plus virilism).
Tissue for light microscopy was Formalin fixed. Sections were stained with hematoxylin- eosin, and for the demonstration of cytoplas- mic glycogen, the PAS procedure with and without prior diastase digestion was employed. Sections from three tumors, some of whose cells contained electron-dense cytoplasmic granules by electron microscopy, were also stained with the Grimelius technique. Tissue for electron microscopy was fixed in 2% buffered glutaraldehyde and embedded in Epon. Thin sections were stained with lead citrate-uranyl acetate.
The 24-hour urinary hydroxysteroids, 17- ketosteroids, and cortisol levels were measured by the methods of Peterson et al., Green,6 and Farmer et al.,7 respectively.
The tumors were accepted as carcinomas and included in our series only if they fulfilled the criteria cited by Ibanez,8 specifically, mitotic activity, pleomorphism, necrosis, in-
This investigation was supported in part by grant NCI- CA-05831 awarded by the National Cancer Institute.
vasion of retroperitoneal soft tissues, or distant metastases.
Lesser amounts of glycogen were present in cells of two other tumors.
RESULTS
Light Microscopy
The cell shape was round or polygonal in all tumors, both primary and metastatic. Typ- ically, a single central nucleus was surrounded by a moderate amount of pale cytoplasm, and adjacent cells tended to be closely apposed, forming diffuse sheets. Architectural organiza- tion such as cord or acinar formation was not seen. In two tumors, considerable quan- tities of glycogen were present in the primary neoplasm, and in one of these (the patient from whom four specimens were received over a period of several years), glycogen in- creased in quantity and was so plentiful in the final accession, a brain metastasis, that the cells had a clear cell appearance by light microscopy.
Electron Microscopy
In 10 tumors, the cytoplasm of most of the cells contained numerous organelles. A moder- ate number were present in 8 cases, and in the remaining 4, they were scanty.
Mitochondria were round, oval, or elongated and varied in size and shape. Neither crystalline inclusions nor giant mitochondria were seen. An occasional mitochondrion with tubular cristae was found in every case, but they were present in a majority of the cells in only 13 tumors and they invariably coexisted in the same tumor and even in the same cell with mitochondria having typical shelflike cristae. The density of the mitochondrial matrix varied considerably. Large mitochondrial matrical granules (Fig. 1), measuring up to 300 nm, were
present in several cases. They were confined to scattered cells and to occasional mitochondria within these cells. A single granule was present and it was round and uniformly electron dense. There was no relationship between the presence of these matrical granules and the shape of the mitochondrial cristae.
In every tumor, flattened cisternae of ribo- some-bearing endoplasmic reticulum were seen, frequently forming stacks of up to 10 short, straight, parallel lamellae. A Golgi complex was readily found but was never large. Smooth endoplasmic reticulum was plentiful only in cells of three of the tumors. (Fig. 2), and it formed an anastomosing network of slender tubules throughout the cytoplasm. In a number of the other tumors, smaller amounts of agranu- lar reticulum were present in some of the tumor cells. Lipid vacuoles were present in significant numbers in only nine of the carcinomas. A few lysosomes were found in cells of most of the tumors but they were never numerous.
Nuclear profiles were typically smooth and
round, but in 2 cases, many were indented. Small clumps of heterochromatin were the rule and nucleoli were generally small. In 9 tumors, short, peripheral microvilli were sand- wiched between adjacent cells. In the others, cell membranes were smooth. Acini were never formed. Cell junctions could be found in every tumor, but they were usually confined to small thickenings of apposed cell membranes with few or no tonofilaments. Fenestration of the endothelium of small blood vessels was evident in only 7 carcinomas.
Dense-core, membrane-bound granules mea- suring 300-500 nm were present in the cyto- plasm of a few cells of three carcinomas (Fig. 3). These were nonfunctioning tumors.
In comparing specimens obtained from the same patient over a period of time, altera- tions in the fine structure of the tumor cells included a reduction in the number of orga- nelles, diminution or loss of smooth endoplas- mic reticulum and tubular mitochondrial cristae, an increase in the number of cytoplas-
mic lipid droplets, and in 2 of the 3 cases, accumulation of glycogen (Fig. 4).
DISCUSSION
When adrenocortical carcinomas are present in metastatic locations, they can readily be confused with other carcinomas by light microscopy. Renal cell carcinoma, liver cell carcinoma, and melanoma head the differen- tial diagnosis. The findings from the present study confirm that ultrastructural examina- tion may be helpful in indicating or confirm- ing the diagnosis, but the pathologist must be aware that a considerable range of fine structure may be observed in cells of adreno- cortical carcinomas. We have encountered situations in which a diagnosis of metastatic adrenocortical carcinoma had not been con- sidered by light microscopy and was established only from the ultrastructural findings. It may, however, be difficult or impossible to distin- guish between renal cell carcinoma and adreno-
cortical carcinoma, even at the ultrastructural level.
The number of mitochondria in the tumor cells varies considerably, but in most cells they are present in moderate numbers. They vary in size and shape and are often elongated. Fre- quent mitochondria with tubular cristae were present in fewer than half of the tumors, and in these cases, many cells did not contain mito- chondria of this type. A curious finding was the presence of dense matrical granules up to 300 nm in diameter in some mitochondria.2,9 They are considerably larger than the well- known small mitochondrial matrical granules.
Tubules of smooth endoplasmic reticulum were plentiful in only a minority of the cases. The smooth ER is known to be the locus of enzymes involved in the synthesis of steroid hormones,7 and it was therefore anticipated that carcinomas in which these organelles were abundant might be more likely to possess func- tional activity than tumors whose cells did not possess significant quantities. This did not
prove to be the case. The 3 tumors in which the smooth ER was particularly copious were clinically nonfunctioning. Rough ER was present in every case and tended to form short stacks of lamellae, a feature common to other endocrine tumors. The presence of lipid vac- uoles was noted in a number of the cases. It has been suggested that lipid-rich adrenocor- tical tumors may be less aggressive in their behavior.10 Frequent fat vacuoles have also been described in nonfunctioning adenomas.2 Reduction in the quantity of lipid in adreno- cortical cells has also been noted following the administration of ACTH.3,11
The presence of glycogen in cells of adrenal tumors does not appear to have been pre- viously described. Increase in glycogen has been reported in normal human adrenal cortex cells12 and in the adrenal cortex of rats that have received corticosterone.13 Glycogen in adrenocortical carcinomas is not a frequent occurrence, since it was found in only 4 of the
22 carcinomas in our series, but the pathologist should be aware that it can occur because the tumors assume a clear cell appearance and can readily be mistaken for renal cell carcinoma. As has already been observed, it may not be possible to determine whether a metastatic carcinoma whose cells contain both glycogen and lipid is of renal cell or adrenocortical origin.
The presence of dense-core, membrane- bound granules in the cytoplasm of some cells of three of the carcinomas is puzzling. The Grimelius stain for argyrophilic granules was negative so they may be lysosomes. 1,12,14 Similar bodies seen in a fetal adrenal cortex of 10 weeks’ gestation were called lysosome- like granules. 15 They have also been described in the cytoplasm of testicular interstitial cells in animals. 16
Reddy, Reddy, and Svoboda demonstrated the presence of similar granules in the inter- stitial cells of rodent testes and discussed their
| Case | Function | SER | Mitochondria | |||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Cortisol | Number | Tubular cristae | Large matrical granules | |||||||
| Sex | 17-OH | 17-KS | AM | PM | ||||||
| 1. | M | 3.1 | 3.9 | 15 | 5 | ++ | + | + | ||
| 2. | F | 8.7 | 8 | 20 | 12 | + - | + | + | + | |
| 3. | F | 10 | 18 | 22 | 14 | + | ||||
| 4. | M | 2.9 | 6.6 | * | · | + | ||||
| 5. | M | 8 | 12 | 23 | 10 | + | ||||
| 6. | F | 7.3 | 5.1 | 23 | 12 | + + | + | |||
| 7. | F | 6 | 14 | 21 | 8 | + | + | + | ||
| 8. | M | * | * | 15 | 7 | |||||
| 9. | F | · | · | 19 | 12 | + | + | + | ||
| 10. | F | 61 | 64 | 47 | 45 | Cushing's | + | + | + | + |
| 11. | M | · | * | 25 | 11 | + | + | |||
| 12. | M | 10 | 12 | 23 | 9 | + | + | + | ||
| 13. | F | 11 | 35 | 35 | 38 | Virilism & Cushing's | + | + | + | + |
| 14. | F | · | · | · | * | Cushing's | + | + | ||
| 15. | F | 15 | 45 | 40 | 35 | Virilism | + | + | + | |
| 16. | M | 5 | 6 | 16 | 8 | + | + | |||
| 17. | M | * | · | 25 | 15 | + | + | |||
| 18. | M | 3.5 | 14 | * | * | + | + | + | ||
| 19. | F | 6 | 14 | 18 | 10 | + | + | |||
| 20. | M | · | · | * | · | + | ++ | + | ||
| 21. | F | 36 | 22 | 42 | 40 | Cushing's | ||||
| 22. | M | 9 | 8 | 20 | 9 | + | + | + | ||
| Normal Values: | 17-KS = M 9-22 µg/24 hr (urine) F 6-15 µg/24 hr (urine) 17-OH = M 3-10 µg/24 hr (urine) | Cortisol: | AM = 12 µg% | (plasma) | ||||||
| PM = 5.5 | µg% (plasma) | |||||||||
| F 2-6 ug/24 hr (urine) | ||||||||||
17-OH, 17-hydroxysteroids; 17-KS, 17-ketosteroids; SER, smooth endoplasmic reticulum. Data not available.
relationship to cholesterol and androgen bio- synthesis.17,18 These granules have been de- scribed in the so-called adrenal corticomedul- lary cells. 19,20
It has been suggested that endothelial fenes- trations are scanty in adrenocortical carcino- mas,10 despite their presence in vessels of the normal adrenal cortex. They were present in seven carcinomas in our series, all clinically nonfunctioning.
Correlation between structure and function was not evident in our study (Table 1). Only 5 of the patients with carcinomas had clinical evidence of hormonal hypersecretion, although other tumors could have been forming an in- active hormone.
It is possible that some of the ultrastructural features we describe may have been induced by therapy, but aside from the accumulation of glycogen in 2 tumors (both patients had received chemotherapy), there were no appar- ent differences between the untreated and the treated cases in our series.
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16. Christensen HK, Gillim SW: The correlation of fine structure and function in steroid-secreting cells, with emphasis on those of the gonads. In: The Gonads, edited by KW Kerns, pp. 415-488. New York: Appleton-Century-Crofts, 1969.
17. Reddy J, Svoboda D: Microbodies (peroxisomes) in the interstitial cells of rodent testes. Lab Invest 26(6):657-665, 1972.
18. Reddy JK: Possible properties of microbodies (peroxisomes), microbody proliferation and hypo- lipidemic drugs. J Histochem Cytochem 21(11): 967-971, 1973.
19. Hashida Y, Yunis EJ: Ultrastructure of the adrenal zona glomerulosa in children with reno- vascular hypertension. Hum Pathol 3(3):301-315, 1972.
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Accepted in revised form 3 August 1981.
Request reprints from Elvio G. Silva.