Japan EPS Proceedings
Histopathological Diagnosis and Prognostic Factors in Adrenocortical Carcinoma
Motohiko Aiba, MD and Mariko Fujibayashi, MD
Abstract
A great majority of adrenocortical tumors are benign, and many adrenocortical carcino- mas (ACC) are obviously malignant at presentation. The histopathological diagnosis of ACC is occasionally difficult, particularly with stage I and stage II disease. The prognosis of ACC is generally poor. Surgery is the major treatment, with chemotherapy and radio- therapy being applicable to only restricted patients. The Weiss criteria are useful in diag- nosing the common adult type of ACC. Histopathological prognostic factors of ACC have not been fully established because of the rarity of the disease.
In this article, we first describe the current histopathological diagnostic and prognostic factors of ACC, highlighting the special types of ACC to which Weiss’s criteria are not fully applicable. These special type tumors include pediatric adrenocortical tumors, oncocytomas, and aldosterone-producing tumors of pure zona glomerulosa type. Then we present three cases with unusual small adrenocortical tumors. One patient had an unequivocal ACC showing metastatic disease. One had a histologically defined ACC with no metastasis or macroscopic invasion. The third was a pediatric patient with a tumor showing a nodule-in-nodule pattern with insulin-like growth factor II expression.
Key Words: Adrenocortical carcinoma; histopathological diagnosis; Weiss’ criteria; prog- nostic factors; clear cell/compact cell morphology; functioning tumor; pediatric tumor; cytochrome P450aldo; P45011ß; Insulin-like growth factor II (IGF-II).
Department of Surgical Pathology, Tokyo Women’s Medical University Daini Hospital.
Address correspondence to Dr. Motohiko Aiba, Department of Surgical Pathology, Tokyo Women’s Medical University Daini Hospital, 2-1-10 Nishiogu Arakawa-ku, Tokyo 116-8567, Japan. E-mail: aibasp@ dnh.twmu.ac.jp
Endocrine Pathology, vol. 16, no. 1, 13-22, Spring 2005 @ Copyright 2005 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/05/16:13-22/ $30.00
Introduction
Adrenocortical carcinoma (ACC) occurs rarely with an estimated incidence between 0.5 and 2 per 106 patients in the United States. It has a bimodal age incidence, in young children before age of 5 yr and in adults at ages between 40 and 50 yr [1]. The prognosis of ACC is poor because of advanced stages at diagnosis or surgery (21% in stage I, 20% in stage II, 20% in stage III, 39% in stage IV [2]), a relatively large tumor size at surgery (an average/ median size is about 10 cm or more, and weight is several hundred grams [1-7]), and a relative resistance to chemotherapy and radiotherapy. Mitotane (o,p’-DDD;
1,1-dichlorodiphenyldichloroethane) blocks 11ß-hydroxylase, has adrenolytic actions, and is an antagonist of P-glyco- protein, which is expressed by the adrenal cortex and ACC [8-10]. At the same time, mitotane is an analog of DDT (dichloro- diphenyltrichloroethane), an insecticide and an endocrine-disrupting chemical, that can cause profound side effects.
Diagnostic criteria for ACC have previ- ously been proposed and evaluated [5- 7,11,12]. However, a differential diagnosis of ACC and adenoma is sometimes diffi- cult, particularly in young children and adults with stage I and II diseases [13-17]. The histopathological prognostic factors of ACC are also difficult to investigate because
of its rarity in occurrence and of advanced stages at diagnosis and surgery. Small ACCs are extremely rare, and they may show some important histopathological proper- ties to be addressed.
In this study, we first describe the histo- pathological diagnosis and prognostic fac- tors of ACC, highlighting some special types of adrenocortical tumors to which the diagnostic/prognostic criteria are not applicable. We then present three cases of unusual small adrenocortical tumors that exhibited either a high clinical malignancy, only a histological malignancy, or a tumor in progression.
Histopathological Diagnosis and Prognostic Factors of Adrenocortical Carcinoma
Weiss’ criteria are currently the most useful standard for histopathological diag- nosis of ACC [5,6]. A tumor is defined as ACC when it exhibits three or more of the following criteria:
1. Nuclear grade III or IV according to the criteria of Fuhrman et al. for renal cell carcinoma.
2. Mitotic rate six or more per 50 high power fields (HPF) (10 HPF per each slide for five slides).
3. Atypical mitoses.
4. Clear cells of 25% or less.
5. A diffuse architecture pattern of > 1/3.
6. A confluent necrosis.
7. Unequivocal invasion of vein.
8. Invasion of sinusoidal structure.
9. Invasion of tumor capsule.
It is recommended that every histo- pathologist first use Weiss’ criteria when diagnosing adrenocortical malignancy. Then, one should re-evaluate the results in a clinical context including the patient’s age, whether it is a special type of adrenal tumor or not, the functional aspects of the
tumor, and other factors, because some diagnostic criteria for the common type of ACC may not apply to some special sub- types of adrenocortical tumors.
Clear Cell/Compact Cell Morphology
A normal adrenal cortex has a clear cell zone and a compact cell zone in the zona fasciculata and the zona reticularis. Clear cells have abundant cholesterol esters as a steroid precursor. Compact cells have abundant organelles, which include mito- chondria and smooth endoplasmic reticu- lum that are related to steroidogenesis. When severe stresses occur, clear cells trans- form into compact cells in response to an elevated plasma adrenocorticotropic hor- mone (ACTH) level under the stimulated hypothalamus-pituitary-adrenal (HPA) axis [18]. When the HPA axis is suppressed, the adrenal compact cells either transform into clear cells [18] or are eliminated by apoptosis. These facts show that normal adrenal compact cells are ACTH-depen- dent. In cortisol-producing adenomas and carcinomas, neoplastic compact cells avoid clear cell changes or apoptosis in spite of suppressed HPA axis and retain their mor- phology. Thus, compact cell predominance in tumors represents an unequivocal autonomy as well as tumor autonomy in cortisol-production Cushing’s syndrome.
In pure androgen-producing tumors, excess androgen does not have a negative feedback effect on the HPA axis. In addi- tion, zona reticularis, which produces adrenal androgen and being a non-neoplas- tic counterpart of pure virilizing tumors, normally consists of compact type cells, although it can transform into the clear cell type by suppression of the HPA axis, e.g., glucocorticoid administration or acute pituitary infarction. Thus, virilizing adre- nal tumors may be liable to retain the com- pact cell type. With that, compact cell
| Steroid produced | P450aldo | P45011B | 3ßHSD | |
|---|---|---|---|---|
| zG | aldosterone | ++ | - | ++ |
| zP | - | - | ++ | |
| zF | cortisol | - | ++ | ++ |
| ZR | DHEA | - | ++ | - |
| Hybrid cells | Cortisol aldosterone | + | + | ++ |
Fig. 1. Immunohistochemistry for differentiation of adrenal zonation. A panel of cytochrome P450aldo, P45011ß, and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) dif- ferentiates adrenal zonation [19,21]. Zona glomerulosa, producing aldosterone, exclusively expresses P450aldo and is negative for P45011B. Zona fasciculata and zona reticularis express P45011ß, and zona fasciculata exhibits 3ß-HSD but zona reticularis does not. In addition, another zone, i.e., the progenitor zone (zP) is found which expresses 3ß-HSD, but expresses neither P450aldo, a zona glomerulosa marker, nor P45011ß, a zona fasciculata/zona reticularis marker, suggesting a precursor of both zona glomerulosa and zona fasciculata [19,21]. The great majority of aldoster- one-producing tumors express both P450aldo and P45011B, showing a hybrid of zona glomerulosa and zona fasciculata type cells [20,21]. Only a few aldosterone- producing tumor consist of zona glomerulosa type cells (see Fig. 2) [20,21].
predominance in virilizing tumors might not be as valuable as an indicator of autonomy as that in Cushing’s syndrome. In a pediatric adrenal tumor study where the majority of tumors are purely andro- gen-producing, Wieneke et al. [13] found that more than 30% of what they have defined as compact cell predominance could not be served as a predictor of malignancy.
In primary aldosteronism, a great majority of aldosterone-producing tumors are a hybrid of zona glomerulosa and zona fasciculata types, which express both cyto- chrome P450aldo and P45011} [19-21] (Fig. 1). They are capable of producing both aldosterone and cortisol and are responsive to ACTH. They have charac- teristics of the clear cell/compact cell in morphology. In fact, the majority of them are clear cell predominant. However, a few
aldosterone-producing tumors are pure zona glomerulosa type (Fig. 2), showing a histol- ogy somewhat different from clear cells and compact cells [20,21] (Fig. 2). In a benign zona glomerulosa-type adenoma, it could be harmful to count this feature (i.e., clear cell <25%) as one criterion of carcinoma.
Although a great majority of ACC are compact in cellular type, most compact- cell predominant tumors are benign. Some investigators exclude compact cell pre- dominance as a diagnostic and prognostic factor of ACC [3,11,13]. On the other hand, clear cell predominance is hardly a feature of unequivocal ACC, unless ACC is treated with chemotherapy. It might be more preferable to give one minus score to the histology of clear cell predominance in the diagnostic criteria of ACC than to give one score to the histology of an inconspicu- ous clear cell component. We have recently reported a case with a relatively large cor- tisol-producing adenoma histologically resembling AIMAH (ACTH-independent bilateral adrenocortical macronodular hyperplasia) [22,23].
Pediatric Adrenocortical Tumors
Pediatric adrenocortical carcinomas are even rarer than adult ACC. The disease has a biphasic age distribution, i.e., infantile (<5 yr) and adolescent (>10 yr) [13-17]. A great majority of ACC in infants are functional. They cause virilization, show Cushing’s syndrome infrequently, and develop feminization or primary aldoster- onism exceptionally. ACC in infants often has good clinical outcome [13-17] when diagnosed on the basis of the adult crite- ria. Because Weiss’ criteria are established by analyses of adult cases (ages 20-70 yr) [5,6], one should be careful when apply- ing the criteria to pediatric cases. Wieneke et al. concluded that adrenocortical tumors in children are clinically and histologically
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different from those in adults [13] and proposed the macroscopic and micro- scopic criteria for adrenocortical neo- plasms in pediatric patients: (1) tumor weight > 400 g; (2) tumor size >10.5 cm;
(3) tumor extension into periadrenal soft tissues and/or adjacent organs; (4) tumor invasion into the vena cava; (5) venous invasion; (6) capsular invasion; (7) pres- ence of tumor necrosis; (8) >15 mitosis/ 20 HPF; (9) presence of atypical mitotic figures. According to these criteria, malig- nancy is defined. A tumor is regarded as benign with long-term clinical outcome when it meets two or fewer of those crite- ria. It is indeterminate for malignancy when meeting three criteria. And, it is regarded as a tumor portending a poor clinical outcome when it meets four or more criteria [13].
Prognostic Factors Other Than Resectability and Stage Factors
Unresectable tumors or advanced disease stage generally serve as powerful indepen- dent prognostic factors. Harrison et al. evaluated 16 pathologic factors in 46 patients with resectable ACC [3], including eight of the nine Weiss’ criteria (excluding com- pact cell predominance), tumor size, intratumoral hemorrhage, and others, for 5-yr survival. Among them, three were independent prognostic factors, namely: (1) larger tumor size > 12 cm (n = 30; 5-yr survival rate 22% vs 53%, p < 0.05), (2) mitotic count 6/10 HPF (n= 31, 13% vs 53%, p < 0.05), and (3) intratumoral hemorrhage (n = 23; 22% vs 53%, p < 0.05). Patients’ 5-yr survival rate was 83% for those with no risk factor, 42% for those with one risk factor, 33% for those with two, and 0% for those with all three fac- tors (p < 0.02) [3].
Other Features in Adrenocortical Tumors
Oncocytoma
An oncocytoma is usually nonfunction- ing and consists of cells with eosinophilic
swollen cytoplasm filled with abundant mitochondria [24-26]. It does not usually have a clear cell component. Foamy mac- rophages should not be misinterpreted as clear cells. It sometimes shows nuclear aty- pia with hypertrophic nucleus and nucleo- lus as well as bizarre nuclear morphology. An adrenocortical oncocytic tumor thus has a low threshold within the ACC diagnosing process because it already has two of Weiss’ criteria. The majority of reported adrenocortical oncocytic tumors, however, have a benign clinical course [24- 26]. Thus, diagnosing a malignant adreno- cortical oncocytic tumor should be done carefully. Taking Weiss’ criteria into con- sideration, ACC could perhaps be diag- nosed if an oncocytic tumor satisfies three or more Weiss’s criteria in addition to two scores of clear cell inconspicuousness and nuclear features.
Nuclear Grade
Nuclear grading in Weiss’ criteria is defined according to that of renal cell carci- noma (RCC) established by Fuhrman et al. [5,6]. However, nuclear features are some- times subtly different between ACC and RCC and thus establishing a nuclear grad- ing system for adrenocortical tumor itself is more preferable.
Immunohistochemistry
Using the immunohistochemistry of fro- zen sections, we have investigated about 100 cases of normal adrenal glands and those with various adrenal diseases, includ- ing adrenals resected with RCC (i.e., endocrinologically normal adrenals), adrenocortical adenomas and carcinomas causing primary aldosteronism, Cushing’s syndrome, and virilization, nonfunctioning tumors, multiple adrenal adenomas caus- ing solely primary aldosteronism or both
primary aldosteronism and Cushing’s syn- drome, secondary adrenocortical hyperpla- sia (ectopic ACTH-producing tumors or secondary aldosteronism), PPNAD (pri- mary pigmented nodular adrenocortical disease), AIMAH, adrenals from autopsy cases with severe stresses, pituitary infarc- tion, or glucocorticoid administration, and so on. Frozen sections were immunostained for cytochrome P450aldo, P45011B, P450scc, 3ß-hydroxysteroid dehydrogenase (3ß-HSD), and “mitochondria” (Chemicon) using the Envision system (DAKO). As control sections, normal rabbit serum and phosphate-buffered saline were substi- tuted for the primary antibody. 3,3’-Diamino- benzidine was used as a chromogen and hematoxylin for nuclear counterstaining [19,21]. In selected cases, AMeX-treated sections [27] were stained for the same panel of antibodies. The antibodies for P450aldo and P45011ß were presented to us by Dr. Fumiko Mitani from the Depart- ment of Biochemistry, Keio University School of Medicine [28]. In some cases, 3ß-HSD was enzyme histochemically examined [29], instead of immunohis- tochemistry. Some of the results of this investigation are presented in Figs. 1 and 2.
As for formalin-fixed materials, paraf- fin-embedded sections were deparaffinized and immunostained for cytochrome P450scc, mitochondria, synaptophysin, neural cell adhesion molecule (CD56), insulin-like growth factor II (IGF-II), pro- IGF-II, MIB-1, p53, and others, accord- ing to the adrenal pathology types, using the labeled streptavidin-biotin (LSAB; DAKO) method in an autostainer. Endog- enous peroxidase activity was blocked with 3% hydrogen peroxide solution. Antigen retrieval was conducted for some antibod- ies with a sodium citrate buffer pH 6.0, by microwave treatment, in accordance to the manufacturers’ instruction.
Single tumor cell
30 doublings
1 g, 1cm3, 109 cells Smallest clinically detectable mass with or without micrometastasis
Several hundred grams, 10 cm in largest dimension (mean/median weight/size of ACC at diagnosis/surgery)
9 doublings
1 doubling
1 kg, 103 cm3, 1012 cells Maximum mass compatible with life
Case Presentation: Unusual Features of Small Adrenocortical Tumors
Largeness in ACC is common, and is sometimes indicated as a prognostic factor [1,2,16]. A large ACC may mean a late or terminal stage in its natural history (Fig. 3). On the contrary, small ACC are extremely rare, and may show early and clinically important aspects in the ACC histology. In this section, we present three cases of small adrenocortical tumors. One case has an unequivocal ACC with a metastatic dis- ease, one has a histologically defined carci- noma, and one pediatric case has a tumor showing a nodule-in-nodule pattern. The samples obtained from all three cases were examined by the immunohistochemistry after formalin-fixation.
Case 1
An 83-yr-old female patient was found to have a left adrenal tumor during a pre-
operative CT examination for rectal carci- noma. Her rectal carcinoma was resected with lymphadenectomy, and histologically diagnosed as moderately differentiated adenocarcinoma, T2, N0, M0, Stage I. The adrenal tumor, being clinically regarded as a nonfunctioning adenoma, was resected 6 mo later together with a small hepatic nodule, which was clinically diagnosed as metastasis of the rectal carci- noma. The adrenal tumor, 3.5 x 3.0 × 2.3 cm in size, consisted of various histo- logic features (Fig. 4). Tumor cells showed hyperchromatic atypical nuclei with numerous mitotic figures (39/10 HPF) and atypical mitotic figures with high MIB-1 index. The tumor expressed all nine Weiss’ criteria. It had areas in which many tumor cells contained a spironolactone body-like structure. Her hepatic tumor, 1.5 x 1.5 x 1.3 cm in size, also showed histologic fea- tures similar to those of the adrenal tumor (Fig. 4). Both adrenal and hepatic tumors were positive for cytochrome P450scc and negative for “hepatocyte” (OCH1E5, DAKO), showing the origin in the adre- nal cortex (Fig. 4). The marked prolifera- tive activity allowed the discovery of an early metastatic state of the incidentally dis- covered small ACC. A high mitotic activ- ity, for example, >20 mitoses/50 HPF median survival 14 mo vs 58 mo p < 0.02 [5], and 6/10 HPF 5-yr survival 13% vs 53%, p < 0.05 [3], has been regarded as a prognostic factor. Immunohistochemical markers of cell cycle regulation and prolif- eration are not a better predictor of metastasis in ACC when compared with its histopathology [30]. During the postoperative follow-up, several addi- tional small metastatic foci were found in the liver, and the patient died of the metastatic disease 8 mo after the second operation [31].
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Case 2
A 44-yr-old male patient with hyperten- sion and hypokalemia had a right adrenal tumor resected with a clinical diagnosis of aldosterone-producing adenoma. The case was reported at the International Case Conference held in 2001, and all of the five participating pathologists specialized in adrenal pathology diagnosed this aldos-
terone-producing tumor as carcinomatous [32]. The most characteristic features of this tumor were that (1) it was small (2.5 cm in diameter) and (2) the architec- ture of the tumor appeared halfway to forming a so-called perithelial pattern (Fig. 5). A perithelial pattern is almost a confirmative diagnosis of carcinoma among aldosterone-producing tumors, because three of Weiss’ criteria (diffuse architecture, confluent necrosis, and no clear cell component) are inevitably included. On the other hand, a perithelial pattern was not completely formed in this tumor yet since many tumor cells apart from blood vessels were not necrotic but necrobiotic. Even so, it would be reason- able to consider that early distant metasta- sis could occur during or after the early architectural changes of the ACC. The patient thus required careful follow-up.
Case 3
A 1 yr and 6 mo old girl was diagnosed with a left adrenocortical tumor that caused Cushing’s syndrome. The well-encapsu- lated tumor, 4.5 × 4 × 3.5 cm in size, showed a nodule-in-nodule pattern. The inner nodule showed a diffuse pattern and a low ratio of a clear cell component with some nuclear hypertrophy, while the outer nodule showed less atypical features (Fig. 6). In addition, cells in the inner nodule showed insulin-like growth factor II (IGF-II) immunoreactivity, while cells in the outer nodule and non-neoplastic part of the ad- renal cortex did not (Fig. 6). This IGF-II staining was not accompanied by immuno- reactivity of the E-domain of Pro-IGF-II, thus differentiating this tumor from a tumor of non-islet cell tumor hypoglyce- mia (NICTH) [33]. ACC, including pediatric cases, frequently overexpress IGF-II [17,34,35]. In the present case, histologic features and IGF-II over-
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expression may indicate the tumor’s promotion to atypical adenoma if not car- cinoma, as the inner nodule did not sat- isfy Wieneke’s criteria [13]. Moreover, being such a small tumor, it could have an excellent prognosis [16]. The patient has
been well with no recurrence for 6 yr. It is notable that pediatric virilizing adreno- cortical tumor occurs before the zona reticularis develops. Wilkin et al. reported IGF-II overexpression with or without p53 mutation in pediatric ACC, and hypoth- esized that childhood adrenocortical tumors within the first 3 yr of life arise because of “the persistent fetal adrenal cells, triggered by defective apoptosis” [17]. However, the present tumor cells causing Cushing’s syndrome were not morphologi- cally similar to fetal zone cells.
Summary
The diagnostic and prognostic criteria of ACC are difficult to establish because of its very rare incidence. Some pathologi- cal factors, such as clear cell/compact cell morphology are intimately correlated with functional aspects of tumors. Nearly half to the majority of ACC are functional, with the rest being called nonfunctional tumors. Subdivision of these tumors into several categories limits the number of cases for statistical evaluations in research studies. On the other hand, it could be harmful to diagnose adrenocortical tumors of special types on the bases of the same criteria of common ACC. We recommend that ACC be classified into two groups, i.e., common- type ACC and special-type ACC. Within the special-type ACC, infantile adrenocor- tical tumors should be evaluated accord- ing to the criteria established with the
studies of pediatric tumors. Histopatho- logical diagnosis of other extremely rare subtypes, such as oncocytic tumors and zona glomerulosa-type aldosterone-pro- ducing tumors, should be carefully re-evalu- ated in clinical context, the functional aspects of the tumor, adrenal tumor types, as well as other related factors. Weiss’ criteria as first-line histopathologi- cal diagnosis of adrenocortical malignancy
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and prognosis are recommended for adult common-type tumors. Although a great majority of ACC are large in size at the time of diagnosis or surgery, small ACC can also be highly malignant, may exhibit early phenotypic changes including struc- tural and cellular morphology or immuno- histochemical expression, or may show the
tumor progression, necessitating careful histopathologic evaluation of the tumor.
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