ADRENOCORTICAL CARCINOMA IN INFANCY Report of a Case with Immunohistochemical and Ultrastructural Studies
Toshio Nishikawa1, Takeshi Kasajima1, Akira Kajita1,
Mitsunori Yamakawa2, Gengo Mabuchi3, Hideo Orihata3, Toshimitsu Shibata4, and Makoto Nakazawa4
Department of ’ Pathology, 3Surgery, and “the Heart Institute of Japan, Tokyo Women ‘s Medical College, Tokyo
2 Department of Pathology, Yamagata University, Yamagata
A case of adrenocortical carcinoma associated with congenital heart defect in a 6-month-old Japanese girl is reported. A fist-sized tumor was incidentally noted in the right hypochondrium upon admission for cardiac surgery. No clinical endocrinopathy was evident in this case. The resected tumor was encapsulated with smooth surface and no invasion to adjacent tissues or organs was observed. Histologically, the tumor was composed of small cells with granular or clear cytoplasm, and occasional giant cells with single or multiple nuclei. By electron microscopy, the tumor cells showed various nuclear con- tours with distinct nucleoli and had a moderate amount of cytoplasm contain- ing abundant rough endoplasmic reticulum and mitochondria with variable- sized electron-dense granules. Intercellular desmosome-like junctions were observed in some tumor cells. Immunohistochemically, the tumor cells contained granules positive for estriol, progesterone and cortisol. These morphological findings including electron microscopic features suggested that the tumor cells had a malignant character. ACTA PATHOL JPN 38 : 683~691, 1988.
Key words : Adrenocortical carcinoma, Infant, Nonfunctional tumor
Introduction
Adrenocortical carcinoma (ACC) is an uncommon neoplasm, with a frequency reported to be approximately 0.2% among autopsy cases with carcinomas.1 Pediatric cases frequently reveal clinical manifestations of adrenocortical hyperfunction such as Cushing’s syndrome or inappropriate masculinization or feminization, and only 9% of them are without endocrinopathy.2 In this report, we describe an infant case of ACC with no clinical evidence of endocrine abnormality, in which immunohistochemical
and electron microscopic examination proved useful in the diagnostic procedure.
Case Report
A 6-month-old girl was admitted to hospital for cardiac surgery because of congenital heart disease. She had been found to have a heart murmur at the age of 1 month and was diagnosed as having congenital heart disease (double outlet right ventricle). During hospitalization, it was incidentally noticed that she had a fist- sized, non-tender firm tumor in the right hypochondrium. Computed tomographic scan of the abdomen revealed a solid mass near the upper pole of the right kidney. Laboratory data including blood count, serum enzymes, electrolytes and urinalysis were within normal limits. Urinary vanillylmandelic acid was negative and cate- cholamines in the urine and blood were within normal limits except for a slight elevation of noradrenaline. Cortisol or sex hormones in the serum and urine were not examined. Surgical exploration revealed a non-fixed, firm tumor with a smooth surface, which was surrounded by a vascular fibrous capsule, and adjacent to the upper part of the right kidney. There was slight fibrous adhesion between the tumor and inferior surface of the liver. No invasion to adjacent organs or lymph node metastasis was noted. Complete tumor resection was performed. The postoperative course was uneventful and the patient was discharged 4 months after the operation.
Methods
For histological examination, tissue was fixed in 10% formalin and routinely processed for the preparation of paraffin sections. These sections were used for H.E., PAS, elastica-Masson, alcian blue, Sudan III, Sudan black B and silver impregnation staining. Immunoperoxidase staining (peroxidase-anti-peroxidase methods and avidin-biotin-peroxidase complex method4) for keratin, $100 protein, lysozyme, carcinoembryonic antigen (CEA), a-fetoprotein (AFP), &1-antitrypsin and vimentin (Dakopatts, Denmark) was also carried out. Pieces of tissue were fixed in periodate-lysine- paraformaldehyde (PLP) fixative and frozen in OCT compound after a complete rinse with sucrose- containing phosphate-buffered saline. Frozen sections were then stained by the immunoperoxidase indirect method following the procedure of Farr and Nakane5 for estrone (Teikoku Zoki, Japan), estradiol, estriol, testosterone, progesterone, cortisol, ACTH (Cambridge Diagnostic Inc., U.K.), hHCG and calcitonin (Dakopatts). For analysis of staining specificity, control staining was simultaneously carried out as previously described® and also antiserum specificity was checked using the immunodiffusion method and immunoabsorption test on sections during the staining process. Part of the tumor tissue was fixed in 1.25% glutaraldehyde, postfixed in 1% osmium tetroxide, and embedded in epoxy resin for electron microscopy.
Pathological Findings
The surgically removed tumor measured 5.5 x 7.5 cm and was well circumscribed with thick fibrous connective tissue. The tumor was elastic and fragile in parts. The cut surface revealed scattered hemorrhagic foci and variable-sized necrotic lesions, especially in the central portion of the tumor (Fig. 1). Areas of necrosis extended through approximately half of the tumor. Microscopically, the tumor displayed a solid alveolar and medullary structure composed of round to polygonal cells with finely granular eosinophilic or occasionally clear cytoplasm (Figs. 2a, b). Cells with
clear cytoplasm were observed in 6% of tumor cells in high-power fields (HPF). The nuclei of the tumor cells were round or irregularly indented and contained one or two distinct nucleoli. Moreover, scattered giant cells were present and some of them were multinucleated. Mitotic figures were occasionally observed and the mean num- ber of mitoses was 1.13 in 10 HPF. There were various degrees of thickening of the fibrocollagenous stroma accompanied by 1 small blood vessels. Hardly any vascular Fig. 1. Macroscopic picture of surgically resected tumor, showing cut surface with extensive necrosis and some hemorrhagic foci. or capsular invasion of tumor cells was recognized. Electron microscopically, the tumor cells showed oval to round nuclei occasionally with somewhat folded or irreg- ularly indented nuclear membranes. Nuclei contained one or two prominent nucleoli with moderate peripheral chromatin condensation, and some cells contained evenly dispersed euchromatin with intermingled heterochromatin (Fig. 3). The cytoplasm contained numerous organelles, especially well developed mitochondria and endoplas- mic reticulum. Mitochondria were round, oval or elongated and varied in size. Electron-dense granules were occasionally found within the mitochondrial matrix. Rough endoplasmic reticulum was plentiful, and frequently formed stacks of short, straight and parallel lamellae (Fig. 4). Smooth endoplasmic reticulum was also remarkably developed. Dense-core granules with or without an outer halo were present is the cytoplasm, which were mostly small and spherical and occasionally contained vacuoles (Fig. 5). Desmosome-like junctions were present between some of the tumor cells. Tumor cells gave positive reactions to PAS, Sudan III and Sudan black B stains, but no reactions to alcian blue, Grimelius, or Fontana-Masson stains. Tumor cells revealed positive reactivity for estriol (Fig. 6a), were weakly positive for progesterone and cortisol, but were negative for estradiol, estrone, testosterone, ACTH, hHCG and calcitonin. Vimentin was positively stained in some of the cells (Fig. 6b). Reactivity for keratin, S-100 protein, lysozyme, CEA, AFP or &1-antitrypsin was negative.
Discussion
Adrenocortical carcinomas (ACC) in pediatric patients are usually associated with adrenocortical hyperfunction.2 However, about 9% of them are nonfunctional and may be incidentally found as an abdominal tumor.2,7 Histopathological findings may be important for the diagnosis in such cases. Light microscopic histology reveals trabecular profiles, nests or a diffuse growth pattern of large epithelioid cells with granular, eosinophilic or clear cytoplasm accompanied by capillaries.8 Giant cells
2.
2b
3
4
5
with prominent nuclei and cellular polymorphism are often present. Tumor cell necrosis, hemorrhage, vascular or capsular invasion may also be observed. These findings are, however, demonstrated in other kinds of tumor including renal cell carcinoma and hepatocellular carcinoma.9 Adrenal medullary tumors may demon- strate similar histopathologic aspects.10 Furthermore, the distinction between adrenocortical adenoma and ACC is also problematic.11 Hough et al.12 reported that findings of massive necrosis, vascular invasion, frequent mitotic figures or marked pleomorphism may contribute to a diagnosis of malignancy. They described the number of mitoses in 10 HPF as being more than 1 in malignant tumors of the adrenal cortex. In our present case, although vascular or capsular invasion was not observed, the mitotic index of the tumor cells exceeded 1, and extensive necrosis and moderate to severe cellular pleomorphism were demonstrated.
Electron microscopic examination may be helpful in indicating or confirming the diagnosis of ACC in some cases. Tannenbaum13 described that the ultrastructure of ACC may differ significantly from that of adrenal cortical adenoma. Silva et al.14 reported that there were several cases in which diagnosis of ACC was not considered by light microscopy but was established from the ultrastructural findings. The cytoplasm of the tumor cells usually contains numerous organelles.14 Mitochondria vary in size and shape, and are often elongated. The number of mitochondria in ACC is greater than that in its normal, hyperplastic or adenomatous counterparts.13 Marked disruption of the basement membrane, interdigitated plasmalemma and/or
63
6b
irregular nuclear contours with or without nuclear invagination are frequently observed.13 Electron-dense granules up to 300 nm in diameter may be present in some mitochondria.14 The rough endoplasmic reticulum tends to form short stacks of lamellae. Smooth endoplasmic reticulum is often plentiful, especially in functional tumors, but may also be demonstrated in nonfunctional tumors. In the present case, similar findings were observed in the mitochondria, rough endoplasmic reticulum and smooth endoplasmic reticulum. Moreover, nuclear contours were variable in several cells with distinct nucleoli. These findings suggested that the tumor was malignant. Dense-core granules were present in the cytoplasm of some cells of our present case. Since Grimelius staining for argyrophilic granules was negative, they may have contained lysosomes. Silva et al.14 demonstrated the presence of similar granules in nonfunctional ACC, and McNutt et al.15 observed dense-core granules in the fetal adrenal cortex and described lysosome-like granules. Similar granules have also been found in the interstitial cells of the testis of various animals, and their relationship to cholesterol metabolism has been suggested.16,17
Immunohistochemistry may be another diagnostic tool in cases of ACC. Wick et al.9 reported that vimentin was positive in non-trypsinized specimens of all 20 adrenocortical tumors they studied, that none of them were immunoreactive for cytokeratin, epithelial membrane antigen, $100 protein or CEA, as shown in our case, and that converse reactions were demonstrated in other tumors including renal cell carcinoma, hepatocellular carcinoma or malignant melanoma. They concluded that immunohistochemical examination may aid in differential diagnosis between ACC and other histologically similar tumors.
Some of the nonfunctional tumors may be associated with enzymic defects which result in failure to form active hormones.18 Furthermore, tumor cells may produce five to ten times less steroid than normal cells.19 Whatever the mechanism, nonfunc- tional tumors do not produce excess active hormones, such as cortisol, aldosterone, androgens or estrogens. To our knowledge, there are no reports describing immunohis- tochemical studies which have indicated the presence of steroid hormones in nonfunc- tional ACC. In our case, cells positive for estriol, progesterone and cortisol were immunohistochemically demonstrated in the tumor, although unfortunately no analy- sis of serum or urinary steroid hormones was done.
In conclusion, the case of ACC which we have presented here was characterized not only on the basis of light- and electron-microscopic histology but also immuno- histochemical examination including steroid hormones, all of which were useful for establishing a diagnosis.
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