Clinical Research
Pathologic Features and Expression of Insulin-like Growth Factor-2 in Adrenocortical Neoplasms
Lori A. Erickson, MD, Long Jin, MD, Thomas J. Sebo, MD, PHD, Christine Lohse, Bs, V. Shane Pankratz, PHD, Michael L. Kendrick, MD, Jon A. van Heerden, MD, Geoffrey B. Thompson, MD, Clive S. Grant, MD, and Ricardo V. Lloyd, MD, PHD
Abstract
We analyzed a series of adrenocortical neoplasms to compare the clinicopathologic fea- tures and the expression of insulin-like growth factor-2 (IGF-2) in adrenocortical adenomas and carcinomas. IGF-2 is a growth factor commonly expressed in many tumors including adrenal cortical and medullary neoplasms. Formalin-fixed paraffin-embedded tissues from 64 adrenocortical adenomas and 67 adrenocortical carcinomas were analyzed. The carci- nomas were histologically graded from 1 to 4 based on mitotic activity and necrosis. Tumor weight, size, and follow-up information were obtained by chart review. Expres- sion of IGF-2 was detected by immunohistochemistry with the avidin-biotin-peroxidase complex method and a monoclonal antibody against IGF-2. Adrenocortical carcinomas were larger (mean: 13.1 cm, 787 g) than adenomas (mean: 4.2 cm, 52 g) (p < 0.001). In patients with adrenocortical carcinomas, high tumor grade (3 or 4) (p = 0.01) was associ- ated with decreased survival. Expresssion of IGF-2 was higher in adrenocortical carcino- mas than in adenomas (p < 0.001). These results show that tumor size and weight along with expression of IGF-2 protein are useful features to assist in distinguishing between adrenocortical adenomas and carcinomas, and that high tumor grade is a predictor of survival in adrenocortical carcinomas. However, single immunohistochemical markers such as IGF-2 or single histopathologic features cannot by themselves separate adrenocortical adenomas from carcinomas, and a combination of clinical, gross, and microscopic fea- tures are needed to establish the diagnosis in difficult cases.
Key Words: Adrenocortical adenoma; adrenocortical carcinoma; insulin-like growth factor-2.
Departments of Laboratory Medicine and Pathology (LAE, LJ, TJS, RVL), Health Sciences Research (CL, VSP), and Surgery (MLK, JAVH, GBT, CSG) Mayo Clinic and Mayo Foundation, Rochester, Minnesota
Address correspondence to: Dr. Ricardo V. Lloyd, Depart- ment of Laboratory Medicine and Pathology, Hilton 11, Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
Endocrine Pathology, vol. 12, no. 4, 429-435, Winter 2001 @ Copyright 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/98/12:429-435/ $11.75
Introduction
Adrenocortical carcinomas are rare tumors that are associated with a high mortality. Although no single criterion other than metastasis or regional invasion is diagnostic of malignancy in adrenocor- tical neoplasms, a variety of clinical and pathologic criteria have been proposed for diagnosis, grading, and prognosis in adrenocortical neoplasms [1-5]. Clinical features include weight loss, urinary
17-ketosteroid levels, response to adreno- corticotropic hormone stimulation tests, and the presence of Cushing syndrome. Gross features include tumor size and tumor weight. Histologic features included diffuse growth pattern, eosinophilic tumor cell cytoplasm, vascular invasion (venous and/or sinusoidal invasion), necrosis, broad fibrous bands, capsular invasion, mitotic rate, atypical mitotic figures, and nuclear plenomorphism. The systems suggested for
distinguishing benign from malignant adrenocortical neoplasms have sometimes been quite problematic [2-5], which reflects the difficulty in diagnosing some adrenocortical carcinomas.
Recent studies of adrenocortical neo- plasms have suggested that insulin-like growth facotr-2 (IGF-2) may have a role in adrenal tumorigenesis. The IGF-2 gene is located on chromosome 11p15. This gene is maternally imprinted and is transcribed from the paternal allele [6-7]. IGF-2 is involved in the regulation of cell growth and differentiation in the adrenal gland. Adrenocortical cells are a site of secretion and action IGFs including IGF-2 [8], which is more highly expressed in fetal compared with adult adrenal glands [9]. Overexpression of IGF-2 has been associated with somatic overgrowth in a variety of tissues [10], and increased levels of IGF-2 mRNA have been associated with malignancy in adrenocortical tumors [11].
The goals of the present study were to compare the clinicopathologic features and the expression of IGF-2 in adrenocortical adenomas and carcinomas and to assess the prognostic significance of these features in patients with carcinomas.
Materials and Methods
Formalin-fixed paraffin-embedded tis- sues from 64 adrenocortical adenomas and 67 adrenocortical carcinomas were ran- domly selected from patients who under- went adrenal surgery at the Mayo Clinic, Rochester, MN, between 1972 and 1997. Clinical history and follow-up information including mean age, gender, tumor size and weight, metastases, and outcome were obtained by chart review (Table 1). Adreno- cortical carcinomas were histologically
graded from 1 to 4 based on the number of mitotic figures per 50 high-power fields (using an Olympus microscope with a ×400 magnification) and the presence of necrosis by two of the authors (LAE and RVL) independently (Table 2). Differences in grading were resolved by reviewing the slides together.
Immunohistochemistry
All adrenocortical adenomas and carci- nomas were analyzed from expression of IGF-2 protein by immunostaining with the avidin-biotin-peroxidase method as previously reported [12] using a mono- clonal antibody (clone S2F2) at a 1/100 dilution (Upstate Biotechnology, Lake Placid, NY).
Quantitation and Statistics
The distribution of immunoreactivity were analyzed by quantifying cytoplasmic staining in each case by two of the authors (LJ and RVL) without knowledge of the diagnosis or outcome. The results of the IGF-2 protein immunostaining were quantified as 0, 1+,2+, and 3+. Univariate comparisons between carcinomas and adenomas were made using x2 tests and Wilcoxon rank sum tests. Logistic regres- sion was used to determine the clinical, pathologic, and immunohistochemical fea- tures that significantly distinguished car- cinomas from asdenomas in a multivariable setting. The Kaplan-Meier method was used to estimate overall survival for the patients with carcinomas. Univariate and multivariable Cox proportional hazards models were used to assess which clinical, pathologic, and immunohistochemical fea- tures were associated with overall survival.
| Diagnosis | n | Mean age (yr) | Female /male | Mean tumor size (cm) | Mean tumor weight (g) | Metastases | Died of disease |
|---|---|---|---|---|---|---|---|
| Adrenocortical | 64 | 56 (range: | 44/20 | 4.2 (range: | 52 (range: | 0 | 0 |
| adenoma | 17-81) | 0.5-11.5) | 5.6-465) | ||||
| Adrenocortical | 67 | 53 (range: | 34/33 | 13.1 (range: | 787 (range: | 53 | 49 |
| carcinoma | 17-78) | 4.5-28.5) | 56-4470) |
| Grade | Mitosesª | Necrosis |
|---|---|---|
| 1 | </50 hpf | – |
| 2 | 5-20/50 hpf | + |
| 3 | 21-50/50 hpf | + |
| 4 | >50/50 hpf | + |
ahpf, high-power field as defined in Materials and Methods.
Results
Clinical Features/Survival
The clinical features for both groups of patients are summarized in Table 1. Twenty-three of 64 (35.9%) adrenocorti- cal adenomas and 32 of 67 (47.8%) adrenocortical carcinomas were clinically functional tumors. The mean length of follow-up for patients with adrenocortical carcinomas was 3.4 yr (range: 0-20 yr). For the 14 surviving patients with adreno- cortical carcinoma, the mean time for surgery to last follow-up was 8.0 yr (range: 0.6-20 yr). Fifty-three of the 67 (79%) patients with adrenocortical carcinomas died. The estimated overall survival rates for the patients with adrenocortical carcinomas at 1, 5, and 10 yr were 61, 27,8, and 18.5%, respectively.
Pathology Gross
Tumor size was significantly different between adenomas and carcinomas by mul-
tivariable analysis (p<0.001). Adrenocorti- cal carcinomas were larger (median: 12 cm, 510 g) than adenomas (median: 3.5 cm, 30.8 g). Each 1-cm increase in tumor size was associated with a 2.7-fold increase in the odds that the tumor was a carcinoma.
The largest adrenocortical carcinomas in a patient alive without disease at last follow-up measured 21 cm after 2.5 yr of follow-up. Three patients with tumors measuring 15 cm each were also alive with no disease after 7, 8, and 20 yr of follow-up, respectively. The smallest carcinomas in a patient who died of disease measured 4.5 cm. All five patients with carcinomas measuring <4.5 cm were alive without dis- ease or dead without disease at last follow-up. However, tumor size was not a statistically significant prognostic factor among the adrenocortical carcinomas overall.
Microscopy
Figure 1 illustrates the histologic features of the lesions. Of the 67 adrenocortical car- cinomas, 12 (17.9%) were grade 1, 15 (22.4%) were grade 2, 22 (32.8%) were grade 3, and 18 (26.9%) were grade 4. High tumor grade (grades 3 or 4) was associated with decreased survival (Fig. 2). Patients with high-grade (grades 3 or 4) adrenocor- tical carcinomas were twice as likely to die of disease compared with patients with low- grade (grades 1 or 2) tumors (p=0.007)
In 6 of the 67 (11.2%) patients, adreno- cortical carcinomas were composed pre-
A
B
C
D
100
80
60
%
40
Grade 2
Grade 1
20
Grade 3
0
Grade 4
0
1
2
3
4
5
6
7
8
9
10
Years
| IGF-2 expression (Immunohistochemical staining)ª | ||||
| Dignosis | 0 | 1+ | 2+ | 3+ |
| Adenoma | 29 | 31 | 4 | 0 |
| Carcinoma | 5 | 21 | 33 | 8 |
a0, negative: 1+, weak staining; 2+, moderate staining; 3+ strong staining.
dominantly of oncocytic cells. Three of the oncocytic tumors were grade 2 and three were grade 1. Three of these patients died of disease with primary tumors measuring 20 (grade 2), 15 (grade 1), and 14 cm (grade 2), respectively. The two patients alive with disease had tumors measuring 20 (grade 1) and 8 cm (grade 2), respec- tively. The patient alive without disease at last follow-up had a 9-cm tumor (grade 1). Only one of the oncocytic carcinomas (20 cm, grade 1) was clinically functional (feminizing).
In another 6 of the 67 (11.2%) patients, the carcinomas showed prominent myxoid change. Four of these patients died of dis- ease. The four tumors were all grade 3, clinically functional (Cushing), and ranged in size from 9 to 15.6 cm. One patient was alive with disease and had a 19.5-cm, grade 2, clinically nonfunctional tumor. One patient was alive without disease and had a 12-cm, grade 2, clinically nonfunctional tumor.
Immunohistochemistry
Immunohistochemical staining revealed cytoplasmic localization for IGF-2 protein in both adrenocortical adenomas and carcinomas (Fig. 1). The majority of carci- nomas (92.5%) expressed IGF-2 compared with approximately half (54.7%) of the adenomas. The level of IGF-2 protein expression was significantly higher in adrenocorticla carcinomas than adenomas
(p<0.001) (Table 3). Tumors with higher levels (2+ or 3+) of IGF-2 expression com- pared with tumors with low or absent lev- els of (0 or 1+) were greater than seven times more likely to be carcinomas than adenomas (p = 0.081) after adjusting for tumor size. Although high levels of IGF-2 expression were associated with a malig- nant phenotype, high IGF-2 expression was not a statistically significant prognos- tic factor among the carcinomas in a mul- tivariate setting.
Discussion
Expression of IGF-2 protein showed striking differences between adrenocorti- cal adenomas and carcinomas. Most car- cinomas (92.5%), but only about half of the adenomas (54.7%), expressed IGF-2. The majority of adrenocortical carcinomas (41 of 67 cases, 61%) showed high levels (2+ of 3+) of IGF-2 expression by immu- nohistochemistry as compared with only 6% (4 of 64 cases) of adrenocortical adenomas. IGF-2 is a growth factor involved in cell proliferation and differentiation. High levels of IGF-2 have been shown to be be expressed during embryogenesis [9,13,14]. Overexpression of IGF-2 has been associated with somatic overgrowth [10], a variety of human tumors, and tumor syndromes such as Beckwith- Wiedemann syndrom [15]. 11p15 loss of heterozygosity and high IGF-2 mRNA
expression have been associated with malignancy in adrenocortical neoplasms [11,16]. Gicqual et al. [16] evaluated 82 sporadic adrenocortical tumors and found loss of heterozygosity and or overexpression of IGF-2 mRNA in 93.1% (27 of 29) of adrenocortical carcinomas and in only 8.6% (3 of 35) of benign adrenocortical tumors. Thus, high levels of IGF-2 pro- tein of mRNA expression may be useful feature in the diagnonis of adrenocortical neoplasms.
Tumor grade was prognostically signifi- cant among patients with adrenocortical carcinoma. High tumor grade (grades 3 or 4) was associated with decreased survival. Patients with high-grade (grades 3 or 4) tumors were twice as likely to die of dis- ease than patients with low-grade (grades1 or 2) tumors. Our grading scheme was based on mitotic rate and the presence of necrosis. Mitotic rate has been reported to be one of the strongest predictors of clini- cal virulence [4,17]. Both low- and high- grade tumors showed necrosis as well as atypical mitotic figures. Thus, mitotic rate appeared to be the criterion distinguish- ing low-grade tumors from high-grade tumors and therefore the most predic- tive of biologic behavior. Earlier studies have examined cell-cycle proteins such as Ki-67 as a prognostic factor in adrenocor- tical tumors [18]. Ki-67 was found to cor- relate with mitotic counts and clinical outcome but did not provide any advan- tages over a careful analysis of mitotic activity [18]. Tumor size and weight also showed significant differences between adrenocortical adenomas and carcinomas. Adrenocortical carcinomas were larger (median: 12 cm, 510 g) than adenomas (median: 3.5 cm, 30.8 g) (p< 0.001). Each 1-cm increase in tumor size was associated with a 2.7-fold increase in the odds of a tumor being a carcinoma (p < 0.001).
However, large tumor size alone is not suf- ficient for a diagnosis of adrenocortical carcinoma. Thirty percent (19 of 64) of adrenocortical adenomas in our study mea- sured 5 cm or greater. Large adrenocorti- cal neoplasms that have not metastasized have been reported [2]. In our series, the largest adrenocortical carcinoma in a patient alive without disease at last follow- up measured 21 cm after 2.5 yr of follow- up. Three patients with tumors measuring 15 cm each were also alive with no disease after 7, 8, and 20 yr of follow-up, respec- tively. However, large tumor size remains an ominous feature in the evaluation of adrenocortical neoplasms. Although no patient in our study with a tumor <8 cm died of disease, small metastatic adrenocortiacal carcinomas have been reported [3].
Adrenocortical carcinomas composed predominantly of oncocytic cells [19] and adrenocortical carcinomas with myxoid change may pose diagnostic prob- lems [20-22]. In our study, six adreno- cortical carcinomas were composed predominantly of oncocytic cells, and six showed myxoid change. Although the histopathologic diagnosis of these variants may be mor difficult because of their unusual appearances, the biologic behav- ior of these tumors was similar to that of the other carcinomas in the study.
In summary, in addition to tumor size, weight, and histologic features, IGF-2 may be a useful a diagnostic marker, since this protein is expressed at different levels in adrenocortical adenomas and carcinomas. However, single immunohistochemical markers cannot be used to predict the biologic behavior of adrenocortical tumors, and a series of markers along with clinico- pathologic features and careful histologic examination should be used to establish the diagnosis in difficult cases.
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