ORIGINAL
Clinicopathological features, biochemical and molecular markers in 5 patients with adrenocortical carcinoma
Ryuji Kouyama1), Kiichiro Hiraishi1), Toru Sugiyama1), Hajime Izumiyama1), Takanobu Yoshimoto1), Takumi Akashi2), Kazunori Kihara3), Keiko Homma4), Hirotaka Shibata5) and Yukio Hirata1)
1) Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
2) Department of Pathology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
3) Department of Urology, Tokyo Medical and Dental University Graduate School, Tokyo, Japan
4) Central Clinical Laboratories, Keio University Hospital, Tokyo, Japan
5) Department of Internal Medicine, Keio University, School of Medicine, Tokyo, Japan
Abstract. Adrenocortical carcinoma (ACC) is a very rare malignant tumor with poor prognosis. To gain insight into the pathogenic significance of ACC, we studied clinicopathological features and gene expression profile in ACC. We analyzed five ACC cases (two men and three women) with the median age of 45-year-old who underwent adrenalectomy at our institute. Endocrine studies revealed that two cases had subclinical Cushing’s syndrome (SCS) and one with concomitant estrogen-secreting tumor, while the rest of three cases had non-functioning tumors. Analysis of urinary steroids profile by gas chromatography/mass spectrometry showed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone, 17-OH progesterone, dehydroepiandorosterone (DHEA), and 11-deoxycortisol in all five cases. The pathological diagnosis of ACC was based on Weiss’s criteria with its score ≥3. The mean size of the resected tumors was 87 mm and Ki67/MIB1 labeling index, a proliferative marker, was 3 - 27%. Immunohistochemical analysis revealed a disorganized expression of several steroidogenic enzymes, such as 3ß-hydroxysteroid dehydrogenase, 17a-hydroxylase, and DHEA-sulfotransferase. Among several genes determined by RT-PCR, insulin-like growth factor (IGF)-II mRNA was consistently and abundantly expressed in all 5 tumor tissues. Postoperatively, two cases with SCS developed local recurrence and liver metastasis. The present study suggests that the disorganized expression of steroidogenic enzymes and the overexpression of IGF-II by the tumor are hallmarks of ACC, which could be used as biochemical and molecular markers for ACC.
Key words: Adrenocortical carcinoma (ACC), Urinary steroid metabolites, Insulin-like growth factor (IGF)-II, Disorganized steroidogenic enzymes
ADRENOCORTICAL carcinoma (ACC) is a very rare neoplasm (incidence: 1-2/million) [1], which eas- ily metastasizes to liver, lung, and bone, with a poor prognosis. With the recent advances in various imaging procedures, the detection of incidental adrenal mass has become more frequent, of which 2 to 12% are accounted for by ACC [2, 3]. Therefore, it becomes more impor- tant to differentiate ACC from benign tumors such as adrenocortical adenomas (ACA). Functioning ACC
are often associated with Cushing’s syndrome and/or virilization, with high circulating dehydroepiandoros- terone-sulfate (DHEA-S) [1, 4, 5], whereas most ACCs are nonfunctioning and massive. The Weiss’s criteria on the basis of pathological characteristics are widely used for distinguishing malignant from benign adreno- cortical tumors [6], while Ki-67 (MIB-1), a cell pro- liferative marker, and P53, a tumor suppressor gene product, are both used as diagnostic markers for ACC [7]. However, any biochemical and molecular markers most reliable for the diagnosis of ACC have not been fully identified yet.
Therefore, the present study was aimed to study the clinicopathological features in 5 ACC patients diagnosed and treated at our institute by analyzing
urinary steroid metabolites, expression of steroido- genic enzymes by immunohistochemical study, and several gene expression of interest in the tumor tissues by RT-PCR.
Patients and Methods
Patients
This study was approved by the Ethics Committee of Tokyo Medical and Dental University Hospital; informed consent was obtained from each patient before operation.
Twelve patients with adrenal tumors, comprised of 5 ACC and 7 ACA, underwent adrenalectomy at our institute during 2005 - 2009. Pathological diagnosis of ACC was made on the basis of the Weiss’s criteria with more than three out of 9 scores.
Measurements of serum and urinary steroids
Serum cortisol was measured by an enzyme immunoassay (EIA), serum DHEA-S and urinary free cortisol by a solid-phase radioimmunoassay, and urinary 17-KS and 17-OHCS by a colorimetric method, respectively. Urinary steroid metabolites in 24 hr urine samples were analyzed by gas chromatography/mass spectrometry analysis (GC/MS) [8, 9]; the age- and sex-matched values of urinary excretions of steroid metabolites from 39 healthy men aged 18- to 49-year- old, 145 healthy women aged 18- to 49-year-old, and 19 healthy women aged 50- to 59-year-old were used as references.
Immunohistochemistry
Immunohistochemical analysis of major steroidogenic enzymes, including 3ß-hydroxysteroid dehydrogenase (3B-HSD), 17a-hydroxylase (CYP17), 21-hydroxylase (CYP21), and dehydroepiandrosterone-sulfotransferase (DHEA-ST), was performed on formalin-fixed, paraffin- embedded serial sections, by the biotin-streptavidin amplified method as described [10].
Real-time quantitative RT-PCR
Using a real-time quantitative RT-PCR method, we analyzed the gene expression profile of the major steroidogenic enzymes, such as cytochrome P450 (CYP)17, CYP11B1, HSD3B2, and aromatase, and some growth factors of interest, such as insulin-like growth factor (IGF)-II, transforming growth factor (TGF)-ß2, and TGF-ß1 receptor type 1 (TGF-BR1).
| Primers | Sequences | PCR product size |
|---|---|---|
| IGF-II | Forward 5'-GCTGGCAGAGGAGTGTCC-3' Reverse 5'-GGGATTCCCATTGGTGTCT-3' | 111bp |
| TGF-ß2 | Forward 5'-CCAAAGGGTACAATGCCAAC-3' Reverse 5'-CAGATGCTTCTGGATTTATGGTATT-3' | 114bp |
| TGF-ØR1 | Forward 5'-AAATTGCTCGACGATGTTCC-3' Reverse 5'-CATAATAAGGCAGTTGGTAATCTTCA-3' | 60bp |
| HSD3B2 | Forward 5'-CTTGGACAAGGCCTTCAGAC-3' Reverse 5'-TCAAGTACAGTCAGCTTGGTCCT-3' | 78bp |
| CYP17 | Forward 5'-CTATGCTCATCCCCCACAAG-3' Reverse 5'-CCTTGTCCACAGCAAACTCA-3' | 63bp |
| CYP11B1 | Forward 5'-AGAAGCTGCAACAGGTGGAC -3' Reverse 5'-GTTGAAGCGCCATTCAGG-3' | 128bp |
| GAPDH | Forward 5'-GCTGAGAACGGGAAGCTTGT-3' Reverse 5'-TCTCCATGGTGGTGAAGACG-3' | 136bp |
Total RNA was extracted from the resected tumor tis- sues using RNA zol (GIBCO/BRL, Carlsbad, CA), and cDNA was synthesized using First-Strand cDNA Synthesis Kit (Amersham Pharmacia Biotech Inc., NJ). The PCR primers of the genes were synthesized by Greiner Bio-one (Tokyo, Japan), and their sequences are shown in Table 1. The levels of each mRNA rela- tive to that of glyceraldehyde-3-phosphate dehydroge- nase (GAPDH) were calculated.
Statistical analysis
Date were expressed as means ± SEM. Differences between groups were examined for statistical significance with an unpaired t test. P values less than 0.05 were considered statistically significant.
Results
Clinicopathological characteristics
The clinicopathological features of 5 ACC patients studied are shown in Table 2. Five ACC patients were two men and three women, with the mean age of 45.0 ± 7.6 years. Three had abdominal pain and one with gynecomastia, but two were asymptomatic and found to have adrenal incidentaloma; there were no Cushingoid features in all cases. Seven ACA patients were 2 men and 5 women, with the mean age of 56.6 ± 3.3 years, consisting of 2 overt adrenal Cushing’s syn- drome, 3 subclinical Cushing’s syndrome (SCS), and 2 non-functioning adenomas. Tumor sizes (87.0 ± 9.8
| Case 1 | Case 2 | Case 3 | Case 4 | Case 5 | ||
|---|---|---|---|---|---|---|
| Clinical | ||||||
| Gender (M/F) | (2/3) | M | F | F | M | F |
| Age (year-old) | (45.0±7.6) | 26 | 45 | 35 | 48 | 71 |
| Abdominal pain | (+) | (-) | (-) | (+) | (+) | |
| Other symptoms | gynecomastia | fever | ||||
| Functioning/non-functioning (NF) | SCS, estrogen | NF | NF | SCS | NF | |
| Tumor size (mm) | (87.0±9.8) | 85×70 | 65×60 | 70×55 | 120×70 | 95×70 |
| Tumor laterality | Right | Right | Left | Left | Right | |
| Clinical Stage | II | II | II | III | II | |
| Recurrence/metastases | (+) | (-) | (-) | (+) | (+) | |
| Adjuvant therapy | RT | (-) | (-) | o,p' DDD, chemotherapy | RT | |
| Endocrine | ||||||
| ACTH (pg/mL) | (26.6±12.4) | <5 | 24 | 26 | <5 | 73 |
| Cortisol (ug/dL) | (15.7±2.4) | 19.1 | 8.3 | 11.7 | 19.8 | 19.8 |
| PRA (ng/ml/hr) | (1.6±0.5) | 3.2 | 0.4 | 2.5 | 0.7 | 1.0 |
| PAC (pg/mL) | (111±19) | 95 | 82 | 159 | 64 | 155 |
| DHEA-S (ng/ml) | (3137±1262) | 4567 | 884 | 1504 | 7540 | 1281 |
| Urinary | ||||||
| free cortisol (ug/day) | (65.3±19.7) | 124 | 15.9 | 53.9 | 96.1 | 36.8 |
| 17-KS (mg/day) | (15.5±6.5) | 20.3 | 3.3 | 6.7 | 31.7 | N.D. |
| Pathological | ||||||
| Weiss criteria | (6.4±0.4) | 7 | 5 | 7 | 7 | 6 |
| Ki 67 (%) | (14.8±4.9) | 13 | 3 | 6 | 27 | 25 |
| (mean±SE) |
SCS, subclinical Cushing’s syndrome; RT, radiochemotherapy; N.D., not determined; PRA, plasma renin activity; PAC, plasma aldosterone concentration; DHEA-S, dehydroepiandorosterone- sulfate; 17-KS, 17-ketosteroids
mm) as evaluated by CT scanning were significantly larger than those of ACA (32.3 ± 6.1 mm). As for the tumor laterality, three were on the right adrenal, and two on the left adrenal. Four were stage 2, and one (Case 4) was stage 3. Postoperatively, local recurrences occurred in 2 (Cases 1, 5) who received radiochemo- therapy, and liver metastases occurred in one (Case 4) who had combination of chemotherapy(cisplatin, etoposide) and mitotane(o,p’DDD).
Two ACC patients (Cases 1, 4) presented with SCS who had suppressed plasma ACTH levels (<5 pg/mL), lack of cortisol diurnal rhythm and its non- suppressibility of cortisol to high-dose (8mg) dexame- thasone suppression test (DST), and one (Case 1) with gynecomastia had high estrogen and DHEA-S levels [11]. Serum DHEA-S levels in ACC were significantly elevated (3137 ±1262 ng/ml) compared to those of ACA (155 ± 44 ng/ml). The score of Weiss’s criteria ranged from 5 to 7 with the mean of 6.4 ± 0.4. Ki67
(MIB-1) labeling index was 14.8 ± 4.9%.
Urinary steroid excretion profile
Results of urinary steroid analyses are shown in Table 3 and illustrated in Fig. 1. The increased urinary excretions of the steroid metabolites compared to those of sex- and aged-matched reference values were as follows: increased 16-OH pregnenolone (16HP5), a metabolite of pregnenolone, and pregnenetriol (PT5), a metabolite of 17-OH pregnenolone in all 5 cases, androstenetriol (AT5), a metabolite of DHEA in 4 (Cases 1, 3, 4, 5), reflecting decreased 3ßHSD activity; increased 50-20a pregnanetriol (20a.PT), a metabolite of 17-OH progesterone in all 5 cases, and etiocholanolone (Et), a metabolite of androstenedione in 4 (Cases 1, 3, 4, 5), reflecting decreased 21 hydroxylase activity; increased 50-tetrahydro-11-deoxycortisol (5ßTHS), a metabolite of 11-deoxycortisol in 4 (Cases 1, 2, 4, 5), reflecting decreased 11ß hydroxylase activity.
| Steroid metabolites (µg/g creatinine) | Case 1 | Case 2 | Case 3 | Case 4 | Case 5 | Reference values | ||
|---|---|---|---|---|---|---|---|---|
| (Male) 1 | (Female) 1 | (Female) 2 | ||||||
| Pregnenolone | ||||||||
| 16-OH pregnenolone | 1011 | 62 | 115 | 58 | 1361 | (10 - 55) | (10 - 60) | (9 - 27) |
| Pregnenediol | 8053 | 96 | 245 | 269 | 3900 | (23 - 74) | (22 - 131) | |
| 17-OH pregnenolone | ||||||||
| Pregnenetriol | 35320 | 2754 | 1100 | 1296 | 10112 | (110 - 550) | (118 - 551) | (88 - 222) |
| DHEA | ||||||||
| Androstenetriol | 1028 | 144 | 997 | 1206 | 1574 | (139 - 600) | (122 - 802) | (58 - 274) |
| DHEA | 34833 | 1021 | 1374 | 3726 | 11007 | (88 - 2400) | (47 - 2324) | (56 - 225) |
| 17-OH progesterone | ||||||||
| 5ß-20a pregnanetriol | 2966 | 2459 | 1501 | 661 | 4445 | (230 - 650) | (190 - 1117) | (120 - 384) |
| 5ß-17-OH pregnanolone | 1163 | 620 | 323 | 549 | 588 | (86 - 308) | (50 - 490) | (38 - 118) |
| Androstenedione | ||||||||
| Etiocholanolone | 3378 | 1107 | 2015 | 2147 | 2241 | (440 - 2110) | (582 - 1907) | (290 - 930) |
| Androsterone | 2015 | 2462 | 2600 | 2611 | 2351 | (880 - 3390) | (660 - 2622) | (344 - 956) |
| 11-deoxycortisol | ||||||||
| 5ß-tetrahydro-11-deoxycortisol | 850 | 311 | 131 | 523 | 317 | (20 - 160) | (30 - 149) | (48 - 152) |
Parentheses (reference values for healthy subjects); 1) 5-95% range of healthy male and female, aged between 18- and 49-year- old, 2) 10-90% range of healthy female, aged between 50- and 59-year-old.
Immunohistochemical analysis
By immunohistochemicalanalysis, immunoreactivities of the major steroidogenic enzymes, such as 3ß- hydroxysteroid dehydrogenase (3ßHSD), 17a-hydrox- ylase (P450c17), 21-hydroxylase (P450c21), 11ß- hydroxylase (P450c11), and DHEA-sulfotransferase (DHEA-ST) were detected in the tumor cells to a varying degrees. For example, 3ßHSD immunoreactivities were evident in 2 (Cases 1, 5), but reduced in 3 (Cases 2, 3, 4), whereas P450c17 immunoreactivities were evident in most cases except for one (Case 3). These immunohistochemical features are consistent with the “disorganized” expression of steroidogenic enzymes in individual tumor cells [11, 12].
Gene expression profile
Among several genes of interest as determined by RT-PCR, the expression of IGF -II mRNA was mark- edly increased in all 5 cases, and significantly (p<0.01) greater than in ACA (Fig. 2). The expression of TGF-ß2 mRNA showed no significant difference between ACC and ACA, whereas the expression of TGF-BR1 mRNA in ACC was significantly (p<0.05) less than that in ACA (Fig. 2). Among steroidogenic enzymes genes examined, the expression of HSD3B2 mRNA in ACC
Cholesterol
DHEA-ST
P450scc
DHEA-S
P450c17
P450c17
Pregnenolone(Pr)
17OHPr
DHEA
3 HSD
Pregesterone (P)
170HP
Androstendione
P450c21
17₿HSD
DOC
11-deoxycortisol
Testosterone
P450c11
Corticosterone
P450cl1
P450 arom
P450cl1
Cortisol
E2
Aldosterone
was significantly (p<0.05) less than that in ACA (Fig. 3), while the expression of both CYP17 and CYP11B1 mRNA showed no significant differences between ACC and ACA. The expression of aromatase mRNA increased in only one (Case 1) who showed feminiza- tion symptoms [11].
Relative mRNA Levels/GAPDH
(a)
IGF-II
Relative mRNA Levels/GAPDH
(b)
TGF-ß2
**
10
15
1
10
0.1
0.01
5
0.001
0
ACA
ACC
ACA
ACC
Relative mRNA Levels/GAPDH
(c)
TGF-BR1
*
10
8
6
4
2
0
ACA
ACC
Relative mRNA Levels/GAPDH
(a)
HSD3B2
Relative mRNA Levels/GAPDH
(b)
CYP17
*
15
40
10
30
20
5
10
0
0
ACA
ACC
ACA
ACC
Relative mRNA Levels/GAPDH
(c)
CYP11B1
Relative mRNA Levels/GAPDH
(d)
Aromatase
20
30
15
20
10
5
10
0
0
ACA
ACC
ACA
ACC
Discussion
Among adrenal incidentalomas, it is often difficult to distinguish ACC from benign adenomas [4]. The size of ACC with 4 cm or less account for 2%, 4 to 6 cm for 6%, and greater than 6 cm for 25%, of all adrenal tumors [13, 14]. Two of our 5 cases were incidentally found to have an adrenal mass by imaging studies, while two of our 5 cases had SCS with increased serum DHEA-S levels. Histopathological diagnosis based on Weiss’s criteria is widely used, but sometimes difficult in the case without local invasion or distant metasta- ses. Furthermore, the Ki67 index, an useful marker for proliferative activity, distinguishing malignant from benign tumors [15-17], was greater than 5% in 4 of our 5 cases.
Urinary steroid analysis clearly revealed increased metabolites of corticosteroid precursors, such as 17-OH pregnenolone and 17-OH progesterone in all 5 cases, and pregnenolone, DHEA, 11-deoxycor- tisol, and androstenedione in 4 cases, all of which decreased postoperatively. Despite increased synthe- sis of corticosteroid precursors in ACC regardless of its functional or nonfunctional nature, immunohis- tochemical analysis revealed that not all of the ste- roidogenic enzymes, such as 3ß-HSD, P450c17, and DHEA-ST, are equally expressed in the same tumor cells, but “disorganized” [12]. Urinary steroid anal- yses of our 5 ACC cases showed decreased 3B-HSD activity by increased 16HP5, PT5, and AT5 excretion, decreased 21 hydroxylase activity by increased 20aPT and Et excretion, and decreased 11ß hydroxylase activ- ity by increased 5ßTHS excretion. Our data are con- sistent with those of previous studies [8, 18]. Thus, it is reasonable to consider that the increased urinary metabolites of steroid precursors in ACC are most likely due to the “disorganized” expression of various steroidogenic enzymes by ACC [12]. These data are supported by the immunohistochemical results that two cases presented with SCS showed positive, but weak immunostaining of both 3ßHSD and P450c21 in scattered tumor cells, suggesting the ineffective steroidgenesis by the individual tumor cells. Given the poor prognosis of cortisol-secreting ACC [19], local recurrence and liver metastases occurred in our 2 cases with SCS (Cases 1, 4).
ACC has been associated with genetic abnormalities, such as mutation of p53, reorganization of chromosome 11p15.5 locus, and deletion of ACTH receptor gene
[5]. Loss of heterozygosity (LOH) and overexpres- sion of IGF-II, a paternally expressed imprinted gene located in the 11p15 region, have been observed in most cases with ACC [20-22]. Recently, it has been shown that expression of IGF-II protein was 10-fold greater in ACC than that in ACA and normal adrenal glands [23], and overexpressed in functional ACC [24]. This study clearly demonstrates overexpression of IGF-II gene in ACC compared to that in ACA irrespective of their functional or non-functional nature. It has been also reported that the overexpression of IGF-II and the increased Ki67 index are both useful tools for distinguishing ACC from ACA [16, 25]. It has been shown that IGF-II induces adrenocortical tumor cell proliferation via the IGF-I receptor in vitro [26, 27], suggesting the importance of the IGF-system in adre- nocortical carcinogenesis. Therefore, overexpression of IGF-II could contribute to highly malignant pheno- type of the adrenal tumors. In fact, it has been reported that there was a close relationship between LOH of the 11p15 region and the tumor recurrence, as well as between the overexpression of IGF-II and the clinical stage and prognosis in ACC [28-30]. However, the present study showed no relationships of the overex- pression of IGF-II to the tumor size, Ki67 index, or the clinical staging, although the number of ACC patients in our study was too small to conclude.
A microarray study of adrenocortical tumors showed the upregulated IGF-II cluster genes and the downregulated steroidogenic enzymes cluster genes in ACC compared to those in ACA [31]. Therefore, the increased levels of urinary metabolites of corticosteroid precursors and the overexpression of IGF-II, when combined together, could be useful as biochemical and molecular markers for the diagnosis of ACC.
This study further revealed the decreased gene expression of TGF-BR1, but not TGF-ß2, in ACC, suggesting that the possible involvement of TGF-B in the growth of ACC. Our data are inconsistent with increased expression of TGF-ß2/TGF-BR1 in ACC by microarray study [31]. While TGF-ß inhibits cell growth, it facilitates tumor growth, invasion, and metas- tasis [32]. Therefore, the exact role of TGF-ß signaling pathway in ACC remains to be determined.
The present gene expression profile showed that the expression of HSD3B2 mRNA was significantly lower in ACC than in ACA, while the expression of CYP17 mRNA in ACC was somewhat lower than in ACA, but CYP11B1 mRNA expression was comparable
between ACC and ACA. However, it seems difficult to compare the differential gene expressions of these ste- roidogenic enzymes in such a small number of tumor specimens examined. Thus, further study is necessary to elucidate whether there exist differential expressions of steroidogenic enzymes between benign/malignant and functioning/nonfunctioning adrenocortical tumors in a larger series. Given the steroidogenic enzymes expressed in a “disorganized” manner in each tumor cells, the discrepancy between the gene expression profile of steroidogenic enzymes and that of the uri- nary excretion of steroid metabolites in our study may represent the “heterogeneity” nature of ACC.
In conclusion, the increased urinary metabolites of corticosteroid precursors due to the “disorganized” expression of steroidogenic enzymes, and the overex-
pression of IGF-II in tumor tissues are both hallmarks of ACC, and when combined, they could be useful as biochemical and molecular markers for the diagnosis of ACC.
Acknowledgments
We wish to express our sincere thanks to Dr. H. Sasano, Tohoku University Graduate School of Medicine, for assistance with the immunohistochemi- cal portion of the study. We deeply thank all physicians and nurses who took care of the patients during hospitalization. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and from the Ministry of Health, Labor and Welfare, Japan.
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