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Clinical Research

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N-cadherin in Adrenal Tumors

N-cadherin Expression in Adrenal Tumors: Upregulation in Malignant Pheochromocytoma and Downregulation in Adrenocortical Carcinoma Amir Khorram-Manesh, MD, Håkan Ahlman, MD, PHD, Svante Jansson, MD, PHD, and Ola Nilsson, MD, PHD Abstract Cell adhesion molecules (CAMs) are important regulators of tumor growth. The aim of the present study was to evaluate the expression pattern of CAMs in adrenal tumors regarding origin (cortex vs medulla) and biologic behavior (benign vs malignant). Eightyseven adrenal tumors were investigated by immunocytochemistry (ICC) using monoclonal antibodies against N-cadherin (NCAD), E-cadherin (ECAD), neural cell adhesion molecule (NCAM), and CD44. Western blotting was performed on 30 tumors using the same antibodies. Markers for proliferation (Ki-67) and catecholamine synthesis (tyrosine hydroxylase) were also analyzed in tumors by ICC. NCAD was expressed in 12/27 benign pheochromocytomas (BPCs) (12 familial cases), 8/8 malignant pheochromocytomas (MPCs), 28/30 adrenocortical adenomas, and 9/22 adrenocortical carcinomas. ECAD was expressed in 0/27 BPCs, 0/8 MPCs, 0/30 adrenocortical adenomas, and 2/22 adrenocortical carcinomas. NCAM was expressed in 26/27 BPCs, 7/8 MPCs, 21/30 adrenocortical adenomas, and 17/22 adrenocortical carcinomas. CD44 was expressed in 23/27 BPCs, 6/8 MPCs, 7/30 adrenocortical adenomas, and 4/22 adrenocortical carcinomas. Both cortical and medullary adrenal tumors expressed NCAD, NCAM, and CD44 but were devoid of ECAD. The expression of CD44 and NCAM did not correlate with the malignant potential of tumors. NCAD was upregulated in MPCs, but downregulated in adrenocortical carcinoma. Thus, NCAD appears to be involved in the development of both cortical and medullary adrenal tumors. Key Words: Adhesion molecules; adrenal tumors; N-cadherin; E-cadherin; pheochromocytomas.

Lundberg Laboratory for Cancer Research, Departments of Surgery and Pathology, Sahlgrenska University Hospital, Göteborg, Sweden. Address correspondence to Dr. Amir Khorram-Manesh, Department of Surgery, Sahlgrenska University Hospital, S-413 45 Göteborg, Sweden. E-mail: [email protected] Endocrine Pathology, vol. 13, no. 2, 99–110, Summer 2002 © Copyright 2002 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046–3976/02/13:99–110/ $13.00

Introduction Specific surface proteins, cell adhesion molecules (CAMs), mediate cell adhesion. There are CAMs that require Ca+2 for adhesion (cadherins) and those that do not (e.g., neural cell adhesion molecules [NCAM]). Cadherins (E-, P-, and N) are important for tissue differentiation and structure. During differentiation, the expression of cadherins changes, which affects many aspects of cell-cell adhesion and cell migration. E-cadherin (ECAD)

holds most epithelial cells together. As the ectodermal cells invaginate, they lose their expression of ECAD and start to synthesize N-cadherin (NCAD). In the differentiation of the central nervous system from the neural tube, NCAD gradually becomes the major adhesion molecule. As the neural crest cells begin to migrate, they lose their expression of all cadherins and are thus readily distinguished from the overlying ectoderm (ECAD) and the neural tube (NCAD). Once the neural crest 99

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cells reach their target organs, they differentiate into neurons and will again express NCAD. In the adult organism, expression of ECAD and NCAD is important for the maintenance of tissue structure and integrity; loss of ECAD can be seen in the transition from adenoma to carcinoma and has been suggested to induce invasiveness and metastasis formation [1]. NCAMs appear during morphogenesis as neural epithelial cells fold inward to form the neural crest/tube and maintain neuronneuron adhesion. NCAMs disappear from the neural crest cells during migration to be reinduced during formation of ganglia. In adult animals, loss of NCAM-mediated cell adhesion has been shown to play an important role in the tumorigenesis and metastatic spread of certain tumors [1,2]. CD44, also known as lymphocyte homing receptor, is a hyaluronic acid–binding CAM, present on hematopoietic cells. Expression of CD44 and CD44 variants has been shown to be important for tumor cell migration and metastases [3]. In the present study, we have examined the expression of CAM in adrenal tumors. Three main types of primary adrenal tumors can be identified in adult humans: adrenocortical adenomas (ACAs), adrenocortical carcinomas (ACCs), and pheochromocytomas (PCs) [4,5]. Microscopically, ACAs exhibit clear, lipid-laden cells arranged in sheets or nests. In published series, the majority of surgically treated ACAs were hormonally active (glucocorticoids and mineral corticoids). Nonfunctional ACAs were observed in as many as 5% of adult autopsies [6]. ACCs frequently secrete well-known steroid hormones, their intermediates, or precursors, leading to clinical manifestations in about half of cases. ACCs are microscopically built up by both clear and compact cells and show varying degrees of nuclear pleo-

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morphism. Elevated excretion of 17-ketosteroids, large tumor size (>6 cm), and evidence of locally invasive or distant metastatic disease are features of malignancy [7–10]. PCs arise from the chromaffin cells of the adrenal medulla and are the most common type of adrenal medullary tumor in the adult, usually benign with synthesis of catecholamines (reflected by expression of the rate-limiting enzyme tyrosine hydroxylase [TH]) and chromogranins as carrier. The histologic appearance is highly variable, sometimes with pronounced cellular pleomorphism. Demonstration of distant metastases is the only indisputable criterion of malignancy [11–13]. All PCs are hormonally active and secrete catecholamines and chromogranins as carrier molecules. Adrenal tumors frequently present problems in the assessment of their biologic behavior. Attempts to identify histopathologic markers indicating malignancy in adrenal cortical tumors have been made [5,8–11,14,15], but the clinical outcome of an individual tumor is still difficult to predict. New markers that can distinguish between benign and malignant adrenal tumors are clearly needed [16–22]. CAMs have been shown to play an important role in tumorigenesis and may serve as a prognostic marker for many tumor types [1,2,23,24], but the role of CAM in adrenal tumors has not been studied in detail [25]. The aim of the present study was to evaluate the expression pattern of CAM in adrenal tumors regarding origin (cortex vs medulla) and biologic behavior (benign vs malignant). We chose to study the expression of NCAD, ECAD, NCAM, and CD44 in several tumors because these CAMs have previously been shown to be of great importance in human cancer development.

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Table 1. Characterizations of Primary Antibodies Used for Immunocytochemistry Antigen

Epitope

Typea

Clone

Catalogue

Dilution

TH

Unknown

MC

2/40/15

1017 381

1/150

CD44

Unknown

MC

A3D8

C7923

1/100

ECAD

Unknown

MC

HECD-1

13-1700

1/1000

NCAD

Unknown

MC

3B9

18-0224

1/50

NCAM

Unknown [26]

MC

ERIC-1

SC-106

1/500

66-bp encoded repetitive element [29]

MC

MIB-1

0607

1/50

Ki-67

a

Antigen retrieval Microwave treated, EDTA-NaOH, pH 6.0 Microwave treated, citrate buffer, pH 6.0 Microwave treated, EDTA-NaOH, pH 6.0 Microwave treated, EDTA-NaOH, pH 6.0 Microwave treated, EDTA-NaOH, pH 6.0 Microwave treated, EDTA-NaOH, pH 8.0

Source Boehringer Mannheim GmbH (Germany) Sigma (St. Louis, MO) Zymed (San Francisco, CA) Zymed

Santa Cruz Biotechnology (Santa Cruz, CA) Immunotech (Marseille, France)

MC, mouse monoclonal.

Materials and Methods Tumors

A total of 87 adrenal tumors were investigated by immunocytochemistry (ICC) and/or Western blotting. Benign adrenal tumors (sporadic and familial) were randomly selected among specimens registered at the Department of Pathology, Sahlgrenska University Hospital between 1965 and 1999. All malignant adrenal tumors, PCs, and ACCs, registered at the Department of Pathology between 1979 and 1999 were included. The criterion for malignancy was the presence of distant metastases at follow-up (range: 3–324 mo). Fresh tumor tissues were obtained from patients operated at the Endocrine Surgical Unit, Sahlgrenska University Hospital. The following tumors were investigated by ICC: 27 benign PCs (12 familial PCs, multiple endocrine neoplasia 2A), 8 sporadic malignant PCs, 30 ACAs (24 nonfunctional, 1 Cushing, and 5 Conn syndromes), and 22 ACCs [26,27]. Fresh

tumor tissues available from 30 tumors (10 sporadic benign PCs, 10 ACAs, and 10 ACCs) were analyzed by Western blotting.

Immunocytochemistry

For ICC staining routinely fixed (formalin) and paraffin-embedded material was used. Sections (5 µm) were mounted on positively charged glass slides, deparaffinized, and rehydrated. All sections were subjected to antigen retrieval by microwave treatment. Primary antibody was applied overnight at 4°C (Table 1). Bound antibodies were visualized by indirect immunoperoxidase techniques (Dako EnVision +; Dako). All slides were coded and evaluated by three observers. Immunolabeling was graded as follows: – = 75% of tumor cells positive. The number of Ki-67 positive nuclei and mitotic figures was counted

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Table 2. Summary of ICC Findings in Adrenal Tumorsa

BPCs (n = 27) MPCs (n = 8) ACAs (n = 30) ACCs (n = 22)

TH

CD44

NCAD

ECAD

NCAM

Ki-67 index

27/27 (0/0/0/27) 8/8 (0/0/0/8) 0/30 (30/0/0/0) 0/22 (22/0/0/0)

23/27 (4/6/1/16) 6/8 (2/0/1/5) 7/30 (23/6/1/0) 4/22 (18/1/2/1)

12/27 (15/4/6/2) 8/8 (0/3/1/4) 28/30 (2/5/6/17) 8/22 (13/5/3/1)

0/27 (27/0/0/0) 0/8 (8/0/0/0) 0/30 (30/0/0/0) 2/22 (20/2/0/0)

26/27 (0/2/8/16) 7/8 (1/0/1/6) 21/30 (9/4/8/9) 17/22 (5/4/7/6)

8 ± 12 14 ± 24 11 ± 13 367 ± 780

a

The results for TH, CD44, N-cadherin, E-cadherin, and NCAM are given as the number of positive tumors/total number of tumors. The percentage of labeled cells in positive tumors is given in parentheses (0/+/++/+++). The number of positive cells was graded as follows: 0 = no labeled tumor cells, + = 1–24% positive cells, ++ = 25–75% positive cells, +++ = >75% positive cells. For Ki-67, the number of positive nuclei in 10 HPF is given as the mean ± SD.

Table 3. Expression of CAMs in Adrenal Tumors Studied by Western Blottinga

ACCs ACAs BPCs a

NCAM

ECAD

NCAD

CD44

8/10 9/10 10/10

0/10 0/10 0/10

5/10 9/10 10/10

6/10 9/10 10/10

Values represent the number of positive tumors/total number of tumors investigated.

in 10 high-power fields (HPF) (0.16 mm2) from each tumor. Western Blotting

Frozen tumor tissues were homogenized in 10 mM potassium-phosphate buffer (pH 6.8) containing 1 mM EDTA, 10 mM 3-(3-cholamidopropyl) dimethylammonio1-propane sulfate, 1 µg/mL of aprotinin, 10 µg/mL each of leupeptin and pepstatin, and 1 mg/mL of Pefablock. Homogenates were sonicated twice for 15 s followed by centrifugation for 10 min at 10,000g. The clear supernatant was withdrawn, assayed for protein content according to Bradford, and stored at –80°C. Aliquots of proteins (20–35 µg protein) were diluted in sample buffer. Reducing agents were added followed by denaturation at 70°C for 10 min and electrophoresis on precast polyacrylamide gels (10% NuPAGE BisTris-gels; Novex, San Diego, CA) using NuPAGE 3-(N-morphonolino)propanesulfonic acid (MOPS) sodium dodecyl sulfate as running buffer. Proteins were

transferred to polyvinyldifluoride membranes using a NOVEX blotting system. Membranes were incubated with primary antibodies (Table 1) at 4°C overnight followed by alkaline phosphatase–conjugated goat–anti-mouse antibody and CDP-Star (Tropix, Bedford, MA) as substrate. Membranes were exposed to enhanced chemiluminescence film at room temperature for 10–120 s. Molecular weight markers (See-Blue or Mark 12; Novex) were used to calculate the apparent size of immunoreactive proteins. Results The results of ICC and Western blotting are summarized in Tables 2 and 3. N-cadherin

ICC demonstrated NCAD in the normal adrenal cortex and medulla. The labeling was confined to cell membranes of cortical parenchymal cells and chromaffin cells. Strong labeling was observed in all layers of the adrenal cortex, whereas labeling of the medulla was much weaker (Fig. 1). Twelve of 27 (45%) benign PCs (BPCs), 8 of 8 (100%) malignant PCs (MPCs), 28 of 30 (94%) ACAs, and 9 of 22 (41%) ACCs were positive for NCAD. The labeling was confined to the cell membranes of tumor cells with no staining over

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Fig. 1. Immunocytochemical demonstration of NCAD, NCAM, and CD44 in normal adrenal gland. All antibodies labeled both cortical parenchymal cells and chromaffin cells of the medulla. NCAD labeling was stronger in the cortex, whereas NCAM and CD44 labeling were stronger in the medulla. All antibodies labeled cell membrane, and cytoplasm was only weakly stained.

stroma, blood vessels, or lymphocytes (Fig. 2). In only one adenoma, NCAD staining was predominant in the cytoplasm. Two ACAs with negative staining were both nonfunctional. Western blotting demonstrated a single immunoreactive band of approx 100 kDa in 10 of 10 (100%) investigated BPCs, 9 of 10 (90%) ACAs, and 5 of 10 (50%) ACCs (Fig. 3). The results of ICC and Western blotting were identical except for three ACCs (two were positive by Western blotting but negative by ICC, and one was positive by ICC but negative by Western blotting). E-cadherin

ICC could not demonstrate ECAD in the normal adrenal cortex or medulla. None of 27 BPCs (0%), 8 MPCs (0%), and 30 ACAs (0%) were positive for ECAD. The vast majority of the ACCs was also negative; two of twenty-two ACCs were positive for ECAD with mainly cytoplasmic staining (Fig. 2). Western blotting did not detect ECAD in any of the investigated tumors (0 of 10 PCs, 0 of 10 ACAs,

0 of 10 ACCs). The results of ICC and Western blotting were identical in all tumors except for three highly malignant ACCs (stage IV), which all were positive only by ICC. Neural Cell Adhesion Molecule

ICC demonstrated NCAM in the normal adrenal cortex and medulla. The labeling was confined to cell membranes of both cortical parenchymal cells and chromaffin cells. Strong labeling was observed in the medulla, whereas labeling in all layers of the adrenal cortex was weaker (Fig. 1). Immunocytochemically 26 of 27 (96%) BPCs, 7 of 8 MPCs (88%), 21 of 30 (70%) ACAs, and 17 of 22 (78%) ACCs were positive for NCAM. The labeling was mainly distributed over the cell membranes of tumor cells (Fig. 2). However, a few tumor cells also had a cytoplasmic staining. Western blotting demonstrated double immunoreactive bands of approx 180 and 150 kDa, respectively, in 10 of 10 (100%) investigated BPCs, 9 of 10 (90%) ACAs, and 8 of 10 (80%) ACCs (Fig. 3).

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Fig. 2. Immunocytochemical demonstration of NCAD, NCAM, ECAD, and CD44 in primary adrenal tumors. NCAD, NCAM, and CD44 were expressed in all types of adrenal tumors, usually with a distinct membrane labeling. ECAD was not demonstrated in the majority of adrenal tumors, but a few ACCs demonstrated patchy, cytoplasmic labeling.

The results of ICC were almost identical to those of Western blotting. CD44

ICC demonstrated focal and weak CD44 labeling in cell membranes of cortical parenchymal cells (zona glomerulosa) and strong labeling in cell membranes of chromaffin cells of the medulla (Fig. 1).

Positive CD44 labeling was demonstrated in 23 of 27 (85%) BPCs and 6 of 8 (75%) MPCs, while the figures for ACA and ACC were 7 of 30 (23%) and 4 of 22 (18%), respectively. All PCs had strong membrane staining, while cortical tumors had very weak cytoplasmic staining (Fig. 2). CD44+ lymphocytes were also observed in the stroma. However, the number of positive

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Fig. 3. Western blotting demonstrating NCAD and NCAM and CD44 in adrenal tumors. A single NCAD band of approx 100 kDa was detected in all sporadic BPCs (10 of 10), a majority of ACAs (9 of 10), and in half of the ACCs (5 of 10). NCAM was demonstrated as two immunoreactive bands of approx 150 and 180 kDa, respectively, in 100% of investigated BPCs (10 of 10), 90% (9 of 10) of ACAs, and 80% (8 of 10) of ACCs. CD44 was demonstrated as a single band of approx 65 kDa in 100% of investigated BPCs (10 of 10), 90% of ACAs (9 of 10), and 60% of ACCs (6 of 10).

lymphocytes was low in all tumors examined. Western blotting demonstrated a single immunoreactive band of approx 65 kDa in all tumors studied. Ten of 10 BPCs (100%) and 9 of 10 (90%) ACAs were positive, while only 6 of 10 (60%) ACCs turned out to be positive. The results confirm those seen for BPCs and ACAs using

ICC but differ for ACCs (all stage IV). These four ACCs were positive using Western blotting, but were negative using ICC. Ki-67 and Mitotic Index

The number of Ki-67-positive tumor cells in adrenal tumors was as follows (mean

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± SEM immunopositive cells per 10 HPF, n = number of tumors): BPCs (8 ± 12/ 10 HPF; n = 27), MPCs (14 ± 24/10 HPF; n = 8), ACAs (11 ± 13/10 HPF; n = 30), ACCs (367 ± 780/10 HPF; n = 22). The corresponding figures for mitotic index were as follows: BPCs (0.17 ± 0.38/10 HPF; n = 27), MPCs (0.14 + 0.37/10 HPF; n = 8), ACAs (0.07 ± 0.25/10 HPF; n = 30), ACCs (3.25 ± 2.75/10 HPF; n = 22) (Fig. 2). Tyrosine Hydroxylase

All adrenal tumors were stained with antibody against TH in order to confirm or exclude the medullary origin of the tumors. All BPCs and MPCs, demonstrated strong cytoplasmic staining for TH. No staining was observed in any adrenocortical adenomas (0/30) or adrenocortical carcinomas (0/22). Delicate TH-positive nerve fibers and varicosities were observed in the adrenal cortex. These findings confirm the adrenomedullary origin of all benign and malignant PCs [28]. Discussion CAMs have been shown to be important for tumorigenesis and invasiveness of many tumors [1,2,24]. Assessment of the malignant potential of adrenal tumors is difficult [11–14]. In the present study, we evaluated the expression of CAM in adrenal tumors and their possible role in tumor development. NCAD expression in normal adrenal cortex and medulla has not been thoroughly evaluated [29,30]. In endocrine tumors emanating from pancreatic B-cells, NCAD is not downregulated with the transition from adenoma to carcinoma [1]. In the present study, NCAD was demonstrated in both normal adrenal medulla and cortex. NCAD was immunocytochemically expressed in all types of adre-

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nal tumors studied. The results were confirmed by Western blotting, showing only slightly different expression. Thus, NCAD is expressed in both cortical and medullary tumors regardless of their malignant potential. During cell transformation and tumor progression, NCAD expression seemed to be upregulated in PCs but was downregulated in cortical tumors (Fig. 4). NCAD therefore appears to be involved in the development of malignant tumors of both the adrenal medulla and the adrenal cortex. However, it is not clear why NCAD is upregulated in malignant medullary tumors, but downregulated in adrenal cortical tumors. The downregulation of NCAD in ACCs is consistent with the concept of NCAD being a tumor suppressor and may therefore be of prognostic significance in these tumors. ECAD is not expressed in the adrenal cortex [31]. It has been reported that the loss of ECAD expression coincides with the transition from well-differentiated adenoma to invasive carcinoma in a transgenic mouse model of pancreatic B-cell carcinogenesis [1,2] and during the development of several human epithelial cancers [24]. In the present study, ECAD was not demonstrated in the normal adrenal cortex and medulla, nor could it be detected in the adrenal tumors studied. ECAD is thus not important for normal adrenal gland function or for the development of adrenal tumors. Furthermore, ECAD has no use as a prognostic marker. NCAM is transiently expressed in many tissues during embryogenesis, including epithelial cells. In adult tissue, the expression of NCAM is confined to the nervous system, skeletal muscle cells, and some neuroendocrine organs [2,32]. NCAM expression is observed in a variety of human tumors, including those with neural origin but also those not related to the nervous system [2,23,31]. In the present study,


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Fig. 4. During cell transformation and tumor progression, NCAD expression seemed to be downregulated in ACCs.

NCAM was demonstrated in both the normal adrenal cortex and medulla. Likewise, both adrenocortical and medullary tumors (benign and malignant) expressed NCAM; Western blotting confirmed this result. NCAM is thus expressed in normal adrenal glands. We obtained no evidence for involvement of NCAM in adrenal tumorigenesis, since there was no relation between NCAM expression and malignant phenotype. CD44 has been demonstrated with focal labeling in the cell membrane of cortical parenchymal cells and strong labeling in the cell membranes of chromaffin cells of the adrenal medulla [33]. The possibility of using CD44 as a marker to differentiate ACC from PC has been examined. This analysis revealed morphologic differences between adrenocortical tumors and PCs; greater than 80% of ACAs and almost 70% of ACCs showed weak to moderate cyto-

plasmic staining, but all PCs had strong membrane staining [33]. Our immunocytochemical results confirmed that CD44 was expressed in all types of adrenal tumors; all PCs had strong membrane staining, whereas cortical tumors had very weak cytoplasmic staining. Western blotting confirmed the immunocytochemical results. The role of CD44 in tumorigenesis of adrenal tumors and its importance as a prognostic marker are still unclear. Ki-67 is a proliferation marker, which is detectable in all phases of the cell cycle. Apoptosis appears to play an important role in the maintenance of cell turnover in normal cortex, as well as in tumor cell dynamics. High Ki-67 activity has been reported for ACCs with significantly higher values than seen for ACAs. In other studies, Ki-67 activity was highest in ACCs with metastases. Ki-67 activity had a specificity of 100% and a sensitivity of 50% in determining

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Acknowledgments This work was supported by grants from the Swedish MRC (5520), Swedish Cancer Research Fund (3911), Göteborg Medical Society, The A. Gabrielsson Foundation, Sahlgrenska Hospital Research Funds, B. Uhlander Memorial Fund, and Camilla Landgren Memorial Fund.

References

Fig. 5. Ki-67 activity/10 HPF in different types of adrenal tumors. Ki-67 index was higher in ACCs than in any other type of adrenal tumors. Half of the ACCs had a Ki-67 index higher than 100. FPC, familial PC.

the malignant potential of PCs [34–40]. In our study, the Ki-67 index was higher in ACC than in any other type of adrenal tumor. Half of the ACCs had a Ki-67 index higher than 100 (Fig. 5). For adrenal medullary tumors, Ki-67 staining was expressed in both MPCs and BPCs with no statistical differences. The Ki-67 index thus has a potential to discriminate between malignant and benign cortical tumors. In conclusion, adrenal tumors express specific patterns of CAM, but these patterns do not correlate to the malignant potential. NCAD seemed to be differentially expressed in adrenal cortical and medullary tumors; less than half of the benign PCs displayed NCAD vs all malignant PCs, while almost all ACAs expressed NCAD vs less than half of the ACCs. Downregulation of NCAD in ACC may thus aid as a prognostic marker in adrenal cortical tumors. High expression of the proliferative marker Ki-67 and high mitotic activity in ACC are helpful in distinguishing between malignant and benign adrenocortical tumors.

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