The Glucocorticoid Receptor Is Overexpressed in Malignant Adrenocortical Tumors
Lyndal J. Tacon, Patsy S. Soon, Anthony J. Gill, Angela S. Chou, Adele Clarkson, Johan Botling, Peter L. H. Stalberg, Britt M. Skogseid, Bruce G. Robinson, Stanley B. Sidhu, and Roderick J. Clifton-Bligh
Cancer Genetics Unit (L.J.T., P.S.S., B.G.R., S.B.S., R.J.C .- B.), Hormones and Cancer Group, Kolling Institute of Medical Research, University of Sydney (A.J.G.), Sydney, NSW 2006 Australia; Departments of Endocrinology (L.J.T., B.G.R., R.J.C .- B.), Anatomical Pathology (A.J.G., A.S.C., A.C.), and Endocrine and Oncology Surgery (S.B.S.), Royal North Shore Hospital, Sydney, NSW 2065 Australia; Department of Surgery (P.S.S.), Bankstown Hospital and University of New South Wales, Sydney, NSW 2052 Australia; and Departments of Genetics and Pathology (J.B.), Surgical Sciences (P.L.H.S.), and Medical Sciences (B.M.S.), University Hospital, SE-751 85 Uppsala, Sweden
Context: Adrenocortical carcinoma (ACC) is a rare tumor with a poor prognosis. The Weiss score is the most widely accepted method for distinguishing an ACC from an adrenocortical adenoma (ACA); however, in borderline cases, accurate diagnosis remains problematic. We recently discov- ered that the glucocorticoid receptor (GR) gene NR3C1 is significantly up-regulated in ACCs com- pared with ACAs in global gene expression studies.
Objective: Our objective was to study GR expression in adrenocortical tumors (ACTs) and to assess its utility as an adjunct to the Weiss score.
Design: Microarray analysis, real-time quantitative RT-PCR (qPCR), immunohistochemistry, West- ern blot, and direct sequencing were performed.
Results: Analysis of 28 ACTs by microarray and 49 ACTs by qPCR found NR3C1 expression to be up-regulated in ACCs compared with ACAs (P < 0.001). Western blotting and RT-PCR confirmed the presence of the GRa isoform in ACCs, and no mutations were detected on direct sequencing. Immunohistochemistry for GR in an overlapping cohort of ACTs demonstrated strongly positive nuclear staining in 31 of 33 ACCs (94%), with negative staining in 40 of 41 ACAs (98%) (P < 0.001). This finding was validated in an external cohort of ACTs, such that 14 of 18 ACCs (78%) demon- strated positive nuclear staining whereas 32 of 33 ACAs (94%) were negative (P < 0.001).
Conclusions: The immunohistochemical finding of nuclear GR staining identified ACCs with high diagnostic accuracy. We propose that GR immunohistochemistry may complement the Weiss score in the diagnosis of ACC in cases that display borderline histology. The possibility that GR is tran- scriptionally active in these tumors, and may therefore be a therapeutic target, requires further study. (J Clin Endocrinol Metab 94: 4591-4599, 2009)
A drenocortical carcinoma (ACC) is a rare but highly aggressive malignancy, with an estimated incidence of one to two per million population (1, 2). This is in contrast to benign adrenocortical tumors (ACTs), which occur in at least 3% of the population aged over 50 yr
(1, 3). At present, complete surgical resection of the ma- lignant primary tumor offers the only potential for cure. Accurate histopathological diagnosis is critical to allow appropriate clinical follow-up as well as consideration of adjuvant therapies such as tumor bed irradiation or sys-
Abbreviations: ACA, Adrenocortical adenoma; ACC, adrenocortical carcinoma; ACT, ad- renocortical tumor; GR, glucocorticoid receptor; qPCR, quantitative RT-PCR; TBST, Tris- buffered saline with 0.1% Tween 20.
temic mitotane (4, 5). The Weiss score, a 9-point his- topathological scoring system, is currently the most widely used system for classifying ACTs as benign or malignant (6-8). The original publication from 1984 (7) required the presence of at least four histological criteria for a diagnosis of malignancy. Weiss subsequently reduced the require- ment to three or more criteria in 1989 after some tumors with a score of 3 recurred (8). In 2002, Aubert et al. (9) proposed a weighted system whereby 2 points were awarded for those criteria considered more important (mi- totic rate and eosinophilic cytoplasm), whereas 1 point was awarded for lesser criteria (abnormal mitoses, necro- sis, and capsular invasion). The remaining criteria (nuclear atypia, diffuse architecture, venous invasion, and sinusoi- dal invasion) were excluded. The 1989 Weiss score and Aubert’s 2002 modification are highly correlated, and in either system, a total score of 2 or lower is considered diagnostic of adrenocortical adenoma (ACA), whereas a score of 3 or higher is indicative of malignancy (8, 9). At present, the Weiss score using a threshold of at least 3 for a diagnosis of malignancy is considered the best histolog- ical predictor of malignant behavior (10) and is a highly sensitive predictor of subsequent metastasis. Indeed, there are only isolated case reports of tumors with an initial Weiss score of 2 that proceed to behave in a malignant manner (11). However, the Weiss system is not very spe- cific because the majority of tumors with a Weiss score of 3 are cured by surgery and are thus biologically benign (12-14). A need therefore exists to identify adjuvant test- ing strategies that may further predict the biological be- havior of ACTs, particularly those tumors that are organ confined and that demonstrate equivocal histological fea- tures (Weiss score 3 or 4). Two large recent gene expres- sion studies have confirmed an apparent heterogeneity within ACCs at the molecular level (15, 16). Both groups undertook mRNA microarray gene expression studies of large ACT cohorts and were able to define molecular sig- natures that could differentiate between subtypes of ACC with differing clinical outcomes.
We have also used microarray gene expression profiling to search for molecular markers that may improve diag- nostic accuracy (17). By this method, NR3C1, the gene encoding the glucocorticoid receptor (GR), and MC2R, encoding the ACTH receptor (ACTHR), were identified as differentially expressed in ACCs compared with ACAs.
GR is a ligand-dependent nuclear transcription factor. Two main isoforms, a and B, are produced by the alternate splicing of NR3C1 and differ only in their ligand-binding C-terminal domains (18, 19). GRÆ is a classic ligand-ac- tivated transcription factor, whereas there is evidence that GRØ acts as a dominant-negative regulator of GRÆ sig- naling (20, 21). GR is expressed in most tissues; however,
its role in normal or neoplastic adrenal cortex has not been conclusively determined (22). Several previous studies have demonstrated that MC2R expression is variably re- duced in ACCs (23-25), and increased NR3C1 mRNA has previously been reported in a small subset of functioning ACCs (25). The aim of this study was therefore to evaluate GR expression in ACCs and to assess its diagnostic utility in conjunction with the Weiss score.
Patients and Methods
The primary study was approved by the Northern Sydney Cen- tral Coast Area Health Service Human Research Ethics Com- mittee, Australia. Informed consent was obtained from pa- tients before sample collection. Tumor tissue was surgically removed and snap-frozen in liquid nitrogen and the samples stored at -80 C in the Neuroendocrine Tumor Bank of the Kolling Institute of Medical Research. Approval for immuno- histochemical analysis of an external validation cohort was pro- vided by The University Hospital Human Research Ethics Com- mittee, Uppsala, Sweden. Tumors with a Weiss score lower than 3 were classified as ACAs, whereas those with a Weiss score of 3 or higher were considered to be ACCs.
Microarray analysis was performed on 28 sporadic ACTs (12 ACCs and 16 ACAs) and six normal adrenal cortices. Quanti- tative real-time PCR (qPCR) analysis was performed on an ex- panded cohort of 49 ACTs (17 ACCs and 32 ACAs) and two normal adrenal cortices. Immunohistochemistry of the primary Sydney cohort was performed on 74 ACTs (33 ACCs and 41 ACAs) and four normal adrenals. All tumors available for im- munohistochemistry (n = 74) in the primary Sydney cohort were reviewed independently and assigned a Weiss score by a single pathologist (A.J.G.) who was blinded as to other data (8). In addition, all available slides for the qPCR cohort (n = 46 from 49 total) were reviewed and scored by the same pathologist. Immunohistochemistry of the external validation Uppsala co- hort was performed on 51 ACTs (18 ACCs and 33 ACAs). These tumors were also assigned a Weiss score by a single pathologist (J.B.), blinded to clinical data.
RNA extraction
Total RNA was extracted from fresh-frozen tissue as previ- ously described (26). Only samples with an RNA integrity num- ber (RIN) higher than 7.5 as determined on the Agilent Bioana- lyzer 2100 (Agilent, Santa Clara, CA) were included in the microarray study. A representative sample of the tumor tissue was sent for histological examination, and only sections con- taining at least 80% tumor cells on histology were used for nu- cleic acid extraction.
Microarray analysis
Microarray gene expression profiling was performed on a cohort of 16 ACAs, 12 ACCs, and six normal adrenal cortices using the Affymetrix (Santa Clara, CA) HGU133plus2.0 gene- chip. Details of the analysis have recently been published (17).
qPCR
A cohort of 17 ACCs and 32 ACAs were studied. The ACC cohort included one adrenal bed recurrence because fresh-frozen
tissue was not available from the original tumor. cDNA was generated from 1 µg total RNA using random hexamers (In- vitrogen, San Diego, CA) and the Superscript III first-strand syn- thesis system (Invitrogen) as previously described (26). qPCR was performed in a Rotorgene 3000 (Corbett Research, Mort- lake, New South Wales, Australia) using specific TaqMan gene expression assays (Applied Biosystems, Foster City, CA) and TaqMan Universal PCR Master Mix, NO AmpErase UNG (Ap- plied Biosystems) according to the manufacturer’s instructions. Ribosomal 18S RNA was used as the endogenous control for normalization. The TaqMan assay IDs were as follows: NR3C1, Hs00353740_m1; MC2R, Hs00265039_s1; and 18S RNA, Hs99999901_s1. The gene expression of each tumor was com- pared with a control sample consisting of two pooled normal adrenal cortices. qPCR for each sample were performed in trip- licate and each qPCR experiment repeated. Differences in gene expression were analyzed using REST-XL version 2 (Relative Expression Software Tool) (27) where relative expression ra- tios are computed based on PCR efficiency and crossing-point differences.
RT-PCR isoform analysis
All samples used for real-time qPCR also were assessed for GRa and GRß expression by RT-PCR. Primer pairs were de- signed to span the exon8-exon9@ junction and exon8-exon9B junction, respectively, and the RT-PCR products were visualized on a 4% polyacrylamide gel. The primers and PCR conditions can be found in the supplemental data. Commercial adrenal cDNA (Ambion Inc., Austin, TX) served as a positive control for the GRa, whereas HEK293 cells, which express GRØ (28), were used as a positive control for GRØ analysis.
DNA extraction for genomic NR3C1 sequencing
DNA extraction from 10 fresh-frozen ACC samples was per- formed using standard proteinase K and phenol-chloroform pro- tocols as previously described (26). All eight coding exons of NR3C1 were sequenced, with primer sequences and reaction details supplied in the supplemental material (published as sup- plemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). Sequencing was performed by Sydney University Prince Alfred Macromolecular Analysis Centre (SUPAMAC) using the ABI PRISM 3700 platform (Ap- plied Biosystems).
Immunohistochemistry
Primary Sydney cohort
Immunohistochemistry for the GR was performed on 33 ACCs, 41 ACAs, and four normal adrenal samples. This in- cluded 18 ACCs and 13 ACAs not used in the qPCR analysis. Two of the ACC samples represented adrenal bed recurrences because paraffin slides of the original tumors were unavailable. Immunohistochemistry was performed on formalin-fixed par- affin-embedded tissue sectioned at 4 pm onto positively charged slides (Superfrost plus; Menzel-Glaser, Braunschweig, Ger- many). GR mouse monoclonal antibody (clone 4H2, NCL- GCR; Novocastra, Newcastle-upon-Tyne, UK) was used at a dilution of 1:20. All slides were processed with an automated staining system, the Leica Microsystems BondmaX autostainer (Leica Microsystems, Mount Waverley, Victoria, Australia), used according to the manufacturer’s protocol and with the man-
ufacturer’s retrieval solutions. Heat-induced epitope retrieval was performed for 30 min in the manufacturer’s acidic retrieval solution ER1 (VBS part no. AR9961). A biotin-free detection system was employed (VBS part no. DS 9713). Tonsillar tissue was used as an external positive control for each stain. Slides were scored separately by two independent pathologists (A.J.G. and A.S.C.), blinded to each other’s results and all clinical data. Where there was a discrepancy, a consensus diagnosis was sub- sequently reached by both pathologists using a multiheaded mi- croscope. GR expression was graded semiquantitatively from 0-4+. Only staining of tumor cells was scored, and endothelial cells, stromal cells, and lymphocytes (which were all positive) were disregarded. Completely negative staining was scored as 0. Focal weak staining of nuclei (nuclear blush in 0-10% of cells) was scored as 1+. The presence of more widespread but weak staining of nuclei (staining of 10-50% of tumor cells) was con- sidered 2+. Diffuse weak nuclear staining of most tumor cells (more than 50%) was scored as 3+. Diffuse strong nuclear stain- ing of all tumor cells (more than 95%) was scored as 4+. For binary analysis, a score of 0-1+ was considered negative and 2+-4+ considered positive.
External Uppsala cohort
Immunohistochemistry for the GR was performed on 51 pri- mary ACTs (18 ACCs and 33 ACAs) well matched to the Sydney cohort for clinical and tumor characteristics. The methods dif- fered only in the use of the Techmate autostainer system (Dako, Glostrup, Denmark) for slide processing. The GR scoring was performed independently by P.S. and J.B., blinded as to each other’s results, in the manner detailed above.
Western blot analysis
A representative cohort of 18 functioning and nonfunction- ing ACTs (nine ACCs and nine ACAs) was included in Western blot analysis, selected on the basis of protein availability and quality. Tumor lysates were prepared by homogenization of fresh-frozen tissue in lysis buffer containing 0.1% Triton X-100 (Sigma-Aldrich Corp., St. Louis, MO) and triple protease inhib- itor (Roche Applied Science, Mannheim, Germany). The sam- ples were denatured (70 C for 10 min) before electrophoresis on precast 4-12% bis-Tris gels (Invitrogen). Separated proteins were transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Piscataway, NJ). The membranes were washed in Tris-buffered saline with 0.1% Tween 20 (TBST) and subse- quently blocked in TBST containing 5% (wt/vol) nonfat dry milk powder for 1 h at room temperature. Blots were then probed with GR (clone 4H2, NCL-GCR; Novocastra) at 1:100 overnight at 4C. After washing in TBST, the blots were probed with perox- idase-conjugated goat antirabbit secondary antibody (1:2500) for 1 h at room temperature. The Amersham ECL Plus Western blotting detection reagents (GE Healthcare) were used to visu- alize the detected proteins. Protein loading was normalized to @-tubulin (DM1A; Sigma). Protein lysate derived from H295R cells served as a positive control for GR expression (22).
Cell culture
HEK293 cells (American Type Culture Collection, Manassas, VA; no. CRL-1573) were grown in DMEM (Invitrogen) supple- mented with 10% (vol/vol) fetal calf serum. H295R cells (Amer- ican Type Culture Collection; no.CRL-2128) were grown in DMEM/F12 (Invitrogen) supplemented with 1% ITS+ Premix
| Sydney cohort | Uppsala cohort | |||
|---|---|---|---|---|
| ACC, n = 17 | ACA, n = 32 | ACC, n = 18 | ACA, n = 33 | |
| Sex (male/female)ª | 7/10 | 9/23 | 10/8 | 14/19 |
| Age (yr),ª median (range) | 46 (18-74) | 54 (21-72) | 53 (29-77) | 56 (28-71) |
| Weiss score,b median (range) | 4 (3-9) | 0 (0-2) | 6.5 (3-9) | 0 (0-2) |
| Presentation,ª n (%) | ||||
| Nonfunctioning | 8 (47%) | 14 (44%) | 11 (60%) | 17 (52%) |
| Cortisol excess | 4 (23%) | 7 (22%) | 5 (28%) | 7 (21%) |
| Aldosterone excess | 1 (6%) | 11 (34%) | 1 (6%) | 9 (27%) |
| Virilization | 1 (6%) | 0 | 1 (6%) | 0 |
| Feminization | 1 (6%) | 0 | 0 | 0 |
| Mixed | 1 (6%) | 0 | 0 | 0 |
| Unknown | 1 (6%) | 0 | 0 | 0 |
| Tumor weight“ (g) median (range) | 125 (55-1230) | 23 (7-75) | NA | NA |
| Tumor sizeb (mm), median (range) | 88 (30-220) | 25 (8-50) | 100 (50-190) | 30 (8-65) |
NA, Not available.
a Not statistically significant between ACC and ACA or between cohorts.
b P < 0.001 between ACC and ACA but not significantly different between cohorts.
· P < 0.001 between ACC and ACA.
(BD Biosciences, Bedford, MA) and 5% fetal calf serum. Cell cultures were maintained in a 5% CO2 humidified incubator at 37 C. Cells were harvested in PBS and pelleted. RNA was ex- tracted from HEK293 cells using TRI reagent (Invitrogen), and 1 µg was converted to cDNA to be used in RT-PCR as described above. H295R cells were resuspended in 0.1% Triton X-100 (Sigma-Aldrich) lysis buffer containing triple protease inhibitor (Roche), sonicated for 30 sec, and then pelleted at maximum speed for 5 min. The supernatant was removed and treated as for the tumor lysate for Western immunoblot.
Statistical analysis
Statistical analysis was performed with SPSS version 16.0.1 for Mac (SPSS Inc., Chicago, IL), and P < 0.05 was considered signif- icant. The Mann-Whitney U test was used for qPCR analysis, and immunohistochemistry results were subjected to x2 testing. Spear- man’s correlation coefficient was used to determine the significance of correlations between variables. A K-score was calculated as a measure of agreement between the GR immunohistochemistry scores of the paired pathologists within each ACT cohort. For the microarray data, a univariate analysis performed with affylmGUI analysis suite (39) using a moderated t-statistic, was applied to the normalized data set using the Benjamini-Hochberg correction to identify genes that were significantly differentially expressed be- tween ACCs and ACAs.
Results
Microarray
Univariate analysis of the microarray data found NR3C1 expression to be significantly up-regulated in ACCs compared with ACAs, with a log2 fold change (M-value) of +1.78 and log odds of differential expression (B-statistic) of +14.2. The difference in MC2R expression was less but still significant between ACCs compared with ACAs, with an M-value of -2.28 and B-statistic of +1.81.
qPCR
Validation of the microarray results was performed us- ing qPCR to analyze mRNA levels of NR3C1 and MC2R in 17 ACCs and 32 ACAs. The clinical and pathological characteristics of these tumors and those of the external Uppsala immunohistochemistry cohort are shown in Ta- ble 1. The qPCR analysis confirmed NR3C1 mRNA ex- pression to be significantly up-regulated in ACCs com- pared with ACAs (expression relative to normal ± SEM 2.50 ± 0.31 vs. 0.78 ± 0.07, P < 0.001; Fig. 1A) and MC2R mRNA expression to be significantly down-regu- lated in ACCs compared with ACAs (1.37 ± 0.67 vs. 3.04 ± 0.38, P<0.001; Fig. 1C). Within the ACCs, clin- ically nonfunctioning tumors demonstrated significantly higher NR3C1 expression than did hormone-secreting tu- mors (2.96 ± 0.30 vs. 1.80 ± 0.44, P < 0.05; Fig. 1B); however, a difference in NR3C1 expression was not seen between nonfunctioning and functioning ACAs (0.74 ± 0.12 vs. 0.82 ± 0.09, P = 0.319; Fig. 1B). If the nonfunc- tioning ACCs (n = 8) were excluded from the analysis, a significant difference in NR3C1 was still seen between functioning ACCs (n = 8) and ACAs (1.80 ± 0.44 vs. 0.78 ± 0.07, P <0.005). MC2R expression was higher in functioning ACTs compared with nonfunctioning ACTs (Fig. 1C) and was also higher in functioning ACAs and ACCs relative to their nonfunctioning counterparts (Fig. 1D). The relative expression of NR3C1 was in- versely correlated with MC2R (Spearman’s p-0.475, P = 0.01; see Fig. 2).
GR isoform analysis
RT-PCR demonstrated the expected product size for GRa in all samples, whereas GRØ was not detected in any
A
p<0.001
NS
B
3
3.5
p<0.05
NS
2.5
3
Relative Expression NR3 C1
Relative Expression NR3 C1
2.5
2
2
1.5
1.5
1
T
1
T
T
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ACA
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C
D
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p<0.001
p<0.001
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p=0.001
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Relative Expression MC2R
3.5
Relative Expression MC2R
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3
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of the tumors (data not shown). Sequencing of all eight coding exons of NR3C1 in DNA extracted from 10 ACC samples did not identify any mutations. In Western blot
12
Spearman’s rho -0.475
10
p=0.01
Relative Expression MC2R
8
6
4
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0
0
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2
3
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5
Relative Expression NR3C1
analysis, the expected 94-kDa band consistent with GRa was seen in protein extracted from ACTs. Additional smaller bands were seen in some tumor samples consistent with reported variant N-terminal isoforms of GRÆ (29); a representative blot showing data from six ACCs and three ACAs is shown in Fig. 3.
H295R
ACCs
ACAs
4
100 kDa +
+GR
75 kDa +
50 kDa +
alpha- tubulin
A
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8
Immunohistochemistry
Representative immunohistochemistry images are shown in Fig. 4. Nonneoplastic adrenal tissue demonstrated focal weak staining in the zona glomerulosa and zona reticu- laris, whereas the glucocorticoid-producing zona fascicu- lata was universally negative (Fig. 4E). The normal adre- nal medulla demonstrated strong staining for GR (Fig. 4F). In the primary Sydney cohort, positive nuclear stain- ing for the GR was demonstrated in 31 of 33 ACCs (94%), whereas GR staining was negative in 40 of 41 ACAs (98%). The external Uppsala validation cohort demon- strated positive nuclear staining in 14 of 18 ACCs (78%) and negative GR immunostaining in 32 of 33 ACAs (97%). The distribution of consensus GR scores according
to Weiss score is shown in Fig. 5, and this difference in GR staining between ACCs and ACAs was significant at P < 0.001. The measure of agreement for the classification of GR staining as positive (2+-4+) or neg- ative (0-1) between the two independent pathologists gave a K-score of 0.889 in the Sydney cohort and 1.000 in the Uppsala co- hort. In multivariate analysis, the GR score remained significantly associated with ma- lignancy (defined by Weiss score ≥3) when adjusted for tumor size, weight, and patient age (P < 0.001). A receiver operating char- acteristic curve for GR score against Weiss score gave a calculated area under the curve of 0.969 in the Sydney cohort and 0.843 in the Uppsala cohort.
Discussion
We have demonstrated a strong association of GR overexpression with ACC, a rare and aggressive malignancy with an incompletely understood molecular pathogenesis (30). At present, the Weiss score is routinely used to classify ACTs as benign or malignant. Al- though used widely, this classification system poses challenges when the score is borderline. Currently proposed adjuvant histopathologi- cal markers of malignancy include IGF-II overexpression (31, 32) and the proliferative marker Ki-67 (17, 33-35). The present study suggests that GR overexpression assessed by immunohistochemistry provides additional information that complements the Weiss score and identifies a potential role for this well-known nuclear transcription factor in ad- renocortical tumorigenesis.
The gene encoding GR, NR3C1, was initially selected for further study after microarray analysis revealed a small but highly significant up-regulation of NR3C1 in ACCs compared with ACAs. We used qPCR to confirm the in- crease in NR3C1 mRNA expression in our expanded co- hort of ACCs. Interestingly, the clinically nonfunctioning ACCs showed significantly higher NR3C1 expression than their hormone-secreting counterparts; however, the over- expression of NR3C1 in ACCs compared with ACAs re- mained significant regardless of the functional status of the ACC, and no relationship was seen between NR3C1 expression and functional status in the ACA cohort. Of note, a recently reported global gene expression study of a large adrenocortical tissue cohort has also found NR3C1
GR Score Stratified According to Weiss Score
n=2
n=45
Glucocorticoid Receptor Score
4
0
o
0
3
O
2
· Sydney Cohort o Uppsala Cohort
1
0
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n=72
n=6
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6
7
8
9
Weiss Score
to be modestly (1.458-fold) but significantly (P = 0.0003) up-regulated in ACCs (n = 33) compared with ACAs (n = 22; Gene Expression Omnibus Record GSE10927) (16).
Consistent with previous work (23-25), MC2R expres- sion was variably down-regulated in our cohort of ACCs, although functioning ACCs demonstrated significantly higher MC2R mRNA levels than did nonfunctioning ACCs. A similar pattern of increased MC2R expression in functioning tumors was observed in the benign cohort, demonstrating MC2R expression to be more strongly as- sociated with tumor function and NR3C1 expression to be more significantly associated with malignancy.
Using a commercially available monoclonal antibody specific to the N-terminal domain of the GR, immunohis- tochemical studies confirmed positive nuclear staining for GR in 31 of 33 ACCs (94%), whereas such staining was absent in 40 of 41 ACAs (98%). These findings were cor- roborated in an independent external validation cohort of 51 primary ACTs, with 14 of 18 ACCs (78%) positive for GR immunostaining and 32 of 33 ACAs (97%) negative. Compared with the small albeit highly significant differ- ences in NR3C1 expression detected by microarray and qPCR analysis, these differences in GR expression by im- munohistochemistry were striking. The cause of this ap- parent discrepancy is clear when the positive immunohis- tochemical staining of endothelial cells, stromal cells, and lymphocytes (illustrated in Fig. 4A) is considered. These be- nign nonneoplastic cells, present in both ACCs and ACAs, would have been admixed with neoplastic cells during RNA and protein extraction and thus diluted the apparent differ- ences between tumor groups. A more robust difference be- tween benign and malignant tumors was therefore shown by immunohistochemical detection of GR protein.
Our combined immunohistochemistry cohorts in- cluded 15 ACTs with a Weiss score of 2 or 3. Within this
subgroup, two of eight tumors that demonstrated positive GR immunostaining have recurred, and the affected in- dividuals died, compared with one of seven tumors with negative GR immunostaining. Of note, two Weiss 3 ACCs that did not demonstrate nuclear staining for GR have subsequently had benign clinical courses without recurrence at 84 and 113 months, respectively. The pos- sibility that positive GR immunostaining in an ACT with an intermediate Weiss of 2 or 3 may identify an increased risk of recurrence deserves further study in larger cohorts.
We also note GR immunostaining to be less intense in highly dedifferentiated tumors (see Fig. 5). Of six ACC samples with a Weiss score of 9, two showed negative GR staining, whereas the remaining four samples demon- strated only 2+ positive nuclear GR staining. We interpret this finding to mean either that GR expression may be reduced in poorly differentiated tumors or that technical difficulties may arise with tissue fixation in larger malig- nant samples. Nevertheless, from a purely diagnostic viewpoint, less intense GR staining would not adversely affect the otherwise correct prediction of tumor behavior according to Weiss criteria.
More importantly, the identification of GRa overex- pression in malignant ACTs offers mechanistic insights into carcinogenesis and identifies a potential therapeutic target. Previous investigation of the presence and function of GR in normal or neoplastic adrenal cortex has been limited and inconsistent (22). Before the characterization of the GR protein, dexamethasone-binding sites were de- scribed in rat adrenal glands, the Y-1 mouse adrenocor- tical cancer cell line, and cultured bovine adrenocortical cells (36). A subsequent study reported dexamethasone- binding sites in ACCs and pheochromocytomas but failed to identify such sites in either ACAs or normal human adrenal cortex (37). More recently, GR overexpression has been demonstrated in the nodules of primary pig- mented nodular adrenocortical disease but not in the sur- rounding normal cortex (38). In combination with our results, it appears that GR expression may play a role in the pathogenesis of diverse adrenocortical neoplasms. The further elucidation of GR targets in the adrenal cortex will be important in providing insights into the role of the GR in both the normal and neoplastic adrenal as well as pro- viding a potentially modifiable target in carcinogenesis.
In summary, we have demonstrated increased GRa ex- pression at both the molecular and protein level in ACCs compared with ACAs. The immunohistochemical finding of nuclear GR staining identified ACCs with high diag- nostic accuracy, and this observational data must be stud- ied further to determine its diagnostic, mechanistic, prog- nostic, and therapeutic significance.
Acknowledgments
Address all correspondence and requests for reprints to: Lyndal J. Tacon, Cancer Genetics Unit, Hormones and Cancer Group, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065 Australia. E-mail: ltacon@med.usyd.edu.au.
L.J.T. was supported by an Australian Post-Graduate Award Research Scholarship and a Cancer Institute New South Wales Research Scholars Award. We acknowledge financial support from Leica Microsystems (Mount Waverley, Victoria, Australia) to a value of $500 U.S. in the form of discounted immunohis- tochemical detection kits.
Disclosure Summary: The authors have nothing to disclose.
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