Adrenocortical Carcinoma: A Comprehensive Immunohistochemical Study of 40 Cases
Annikka Weissferdt, MD, FRCPath,* Alexandria Phan, MD,; Saul Suster, MD,¿ and Cesar A. Moran, MD*
Abstract: Adrenocortical carcinomas (ACC) are uncommon tu- mors of the adrenal cortex that are known to follow an aggressive clinical course. The distinction of these tumors from other neo- plasms may sometimes prove difficult due to overlapping clinical, morphologic, and even immunophenotypical features. To this end, we performed a comprehensive immunohistochemical analysis using traditional and novel markers in 40 cases of ACC. An im- munohistochemical panel consisting of 10 traditional and novel antibodies was applied to whole tissue sections of ACC including high-molecular weight cytokeratin (HMWCK), low-molecular weight cytokeratin (CAM5.2), inhibin-a, melan A, chromogranin A, synaptophysin, calretinin, steroid receptor coactivator-1 (SRC-1), Pax8, and Ki67. The percentage of positive tumor cells as well as the intensity of staining were evaluated and scored; for Ki67 the per- centage of positive tumor cells was recorded. Positive staining was observed for SRC-1 (39/40; 97.5%), inhibin-a (37/40; 92.5%), cal- retinin (32/40; 80%), synaptophysin (29/40; 72.5%), melan A (26/40; 65%), and CAM5.2 (9/40; 22.5%). Rare cases showed positivity for chromogranin A (2/40; 5%) and Pax8 (1/40; 2.5%). None of the cases showed any reactivity with HMWCK. The Ki67 index ranged from <5% to 20%. We conclude that there is no single specific marker to reliably distinguish ACC from other primary or meta- static neoplasms. However, a combination of immunohistochemical stains in a panel consisting of SRC-1, inhibin-a, calretinin, and HMWCK may be of aid in the differential diagnosis of these tumors. In addition, Pax8 is only rarely positive in ACC, which is a useful tool in their separation from renal neoplasms.
Key Words: adrenocortical carcinoma, immunohistochemistry, endocrine tumor
(Appl Immunohistochem Mol Morphol 2014;22:24-30)
A drenocortical carcinomas (ACC) are rare tumors accounting for ≤0.2% of all malignancies. Despite multimodal therapies, its prognosis remains poor with a mean survival of <30 months.1-3 These tumors arise
from the cells of the adrenal cortex, specialized steroido- genic cells that produce glucocorticoids, mineralocorticoids, and androgens. Tumorigenesis may lead to abnormal hormone function and result in excess production of steroid hormones that may clinically manifest as Cushing syndrome, Conn syndrome, virilization, or feminization. However, a proportion of ACC may be nonfunctional and patients present with nonspecific symptoms such as ab- dominal pain or fatigue. Because of morphologic variability and histologic overlap, the separation of ACC from other adrenal or retroperitoneal neoplasms may be difficult, es- pecially in patients who do not present with hormonal abnormalities. The initial diagnosis is often based on a small core biopsy providing limited histologic material thereby further complicating diagnostic accuracy. In this scenario, immunohistochemical stains play an important role in the evaluation and differential diagnosis of ACC. In this context, it has to be noted that no single immunohistochemical marker specifically identifies ACC, but that often a wider panel comprising several markers is necessary to arrive at the correct diagnosis.
Herein we present a comprehensive study on the immunohistochemical properties of ACC performed on a large series of cases using an immunohistochemical panel consisting of traditional and novel markers in this field.
MATERIALS AND METHODS
Forty cases of ACC were identified from the path- ology files of the Pathology departments of The University of Houston MD Anderson Cancer Center and the Medical College of Wisconsin. The cases were reviewed and a di- agnosis of ACC was confirmed using the Weiss scoring criteria.4 Representative unstained sections obtained from paraffin blocks were available in all cases to perform im- munohistochemical studies. The sections were incubated with 3% H2O2 in methanol and fetal bovine serum to block endogenous peroxidase activity and nonspecific protein- protein interactions, respectively. Immunostaining was performed using a horseradish peroxidase-labeled polymer system. Tissue sections were incubated with antibodies against high-molecular weight cytokeratin (HMWCK), low-molecular weight cytokeratin (CAM5.2), inhibin-a, melan A, chromogranin A, synaptophysin, calretinin, ste- roid receptor coactivator-1 (SRC-1), Pax8, and Ki67 (Table 1). Diaminobenzidine was used as a chromogen for antigen localization. Adequate positive and negative
Received for publication January 7, 2013; accepted January 30, 2013. From the Departments of *Pathology; ¡ GI Medical Oncology, MD Anderson Cancer Center, Houston, TX; and ¿Department of Patho- logy, Medical College of Wisconsin, Milwaukee, WI.
The authors declare no conflict of interest.
Reprints: Annikka Weissferdt, MD, FRCPath, Department of Patho- logy, MD Anderson Cancer Center, Houston, TX 77030 (e-mail: aweissferdt@doctors.org.uk).
Copyright @ 2013 by Lippincott Williams & Wilkins
| TABLE 1. Specifications of Antibodies Used | ||||
|---|---|---|---|---|
| Antibodies | Company | Clone | Dilution | Antibody Incubation Time (min) |
| HMWCK | Dako, Carpinteria, CA | 34BE12 | 1:50 | 65 |
| CK | BD Biosciences, San Jose, CA | CAM5.2 | 1:50 | 90 |
| Inhibin-a | AbD Serotec, Raleigh, NC | R1 | 1:50 | 65 |
| Melan A | Novocastra, Buffalo Grove, IL | A103 | 1:25 | 60 |
| Chromogranin A | Dako, Carpinteria, CA | DAK-A3 | 1:100 | 60 |
| Synaptophysin | Dako, Carpinteria, CA | SY38 | 1:200 | 65 |
| Calretinin | Cell Marque, Rocklin, CA | Polyclonal | 1:100 | 65 |
| SRC-1 | Cell Signaling, Danvers, MA | 128E7 | 1:100 | 90 |
| Pax8 | Protein Tech, Chicago, IL | 10336-1-AP | 1:100 | 45 |
| Ki67 | Dako, Carpinteria, CA | MIB-1 | 1:100 | 65 |
CK indicates cytokeratin CAM5.2; HMWCK, high-molecular weight cytokeratin; SRC-1, steroid receptor cofactor-1
controls were run for all antibodies tested. The im- munostaining was scored on a sliding scale of 0 to 4+ according to the percentage of reactive cells (0, neg- ative; 1+, 1% to 25%; 2+, 26% to 50%; 3+, 51% to 75%; 4+, 76% to 100%) and subsequently the staining intensity were graded as weak, intermediate, or strong.
RESULTS
Clinical Features
The patients were 18 women and 12 men aged 17 to 76 years with a mean age of 50 years. The main clinical symptoms included hormone-related symptoms (pre- dominantly hypertension and Cushing syndrome) or nonspecific complaints such as abdominal pain, flank pain, or fatigue. Clinical follow-up was available for 26 cases. Twelve patients were alive and well 1 to 181 months
after diagnosis (mean follow-up, 82.3 mo). Fourteen pa- tients had died of their disease 4 to 439 months after diagnosis (mean survival, 59.43 mo). Fourteen patients were lost to follow-up. The patients’ pathologic staging was performed according to the AJCC 7th edition staging system: 1 case was classified as T1, 9 as T1 or T2, 12 as T2, 13 as T3, and 5 as T4.
Macroscopic Features
The tumors were generally large tumors which measured from 4 to 20 cm in size with an average dia- meter of 10cm. The weight ranged from 28 to 3260 g (mean weight, 442 g). The tumors were variably encap- sulated and often surrounded by a thin fibrous capsule. Some tumors invaded through the capsule and into the surrounding fibroadipose tissue. The cut surface revealed pale pink to tan-yellow solid or lobulated tumors. Areas
A
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E
| TABLE 2. Percentage of Immunohistochemically Positive Cells in Adrenocortical Carcinomas | ||||||||
|---|---|---|---|---|---|---|---|---|
| Markers | Staining Pattern | 0 | +1 | +2 | +3 | +4 | Positive Cases | % |
| SRC-1 | Nuclear | 1 | 1 | 0 | 0 | 38 | 39 | 97.5 |
| Inhibin | Cytoplasmic (granular) | 3 | 3 | 6 | 2 | 26 | 37 | 92.5 |
| Calretinin | Cytoplasmic and nuclear | 8 | 8 | 3 | 0 | 21 | 32 | 80 |
| Synaptophysin | Cytoplasmic | 11 | 5 | 3 | 4 | 17 | 29 | 72.5 |
| Melan A | Cytoplasmic | 14 | 7 | 9 | 3 | 7 | 26 | 65 |
| CAM5.2 | Cytoplasmic | 31 | 9 | 0 | 0 | 0 | 9 | 22.5 |
| Chromogranin A | Cytoplasmic | 38 | 0 | 0 | 0 | 2 | 2 | 5 |
| Pax8 | Nuclear | 39 | 0 | 0 | 0 | 1 | 1 | 2.5 |
| HMWCK | Nuclear | 40 | 0 | 0 | 0 | 0 | 0 | 0 |
0 = negative; 1+ = 1% to 25%; 2+ = 26% to 50%; 3+ = 51% to 75%; 4+ = 76% to 100%.
HMWCK indicates high-molecular weight cytokeratin; SRC-1, steroid receptor cofactor-1.
of hemorrhage and/or necrosis were identified in most of the cases.
Histologic Features
Histologically, the tumors consisted of 18 conven- tional ACC, 17 oncocytic ACC, 2 myxoid ACC, 2 rhab- doid ACC, and 1 sarcomatoid ACC. The conventional ACC were characterized by clear to eosinophilic tumor cells growing in a solid, trabecular, or lobulated pattern (Fig. 1A). Deeply eosinophilic cytoplasm with a granular appearance was the hallmark of the oncocytic ACC (Fig. 1B). These cells frequently showed cytologic atypia in the absence of a high number of mitoses. Myxoid ACC showed a prominent myxoid stroma composed of pools, lakes, or microcysts of myxoid or chondroid material (Fig. 1C). Rhabdoid tumors were characteristically com- posed of medium-sized to large tumor cells with eosino- philic cytoplasmic inclusions and large eccentric vesicular nuclei with prominent eosinophilic nucleoli (Fig. 1D). The sarcomatoid ACC was composed of oval-shaped to spindle-shaped tumor cells intermixed with areas of more conventional ACC (Fig. 1E). Hemorrhage and necrosis were present in nearly all cases. The mitotic count ranged from 1 to > 20 mitoses per 10 high power fields.
Immunohistochemical Features
The immunohistochemical features are summarized in Tables 2 and 3. Immunohistochemical stains demon- strated diffuse staining for SRC-1 in almost all cases (39/
| Markers | Weak | Intermediate | Strong |
|---|---|---|---|
| SRC-1 | 15 | 16 | 8 |
| Inhibin | 5 | 10 | 22 |
| Calretinin | 18 | 12 | 2 |
| Synaptophysin | 7 | 7 | 15 |
| Melan A | 11 | 12 | 3 |
| CAM5.2 | 5 | 2 | 2 |
| Chromogranin A | 2 | 0 | 0 |
| Pax8 | 1 | 0 | 0 |
| HMWCK | 0 | 0 | 0 |
HMWCK indicates high-molecular weight cytokeratin; SRC-1, steroid receptor cofactor-1.
40; 97.5%), the majority of which showed intermediate to strong staining intensity in the nuclei of the tumor cells (Fig. 2A). Inhibin-a positivity was seen in a similarly high number of cases (37/40; 92.5%) with intermediate or strong staining intensity in 32 of the 40 cases (Fig. 2B). Although reactive in a great majority of cases, the per- centage of positive cells was more variable for calretinin (32/40; 80%) (Fig. 2C); in addition, this marker often only produced a weak staining signal. Similar ob- servations were made for synaptophysin, a marker that was positive in a large number of cases (29/40; 72.5%), but showed more heterogeneity in terms of percentage of positive cells and staining intensity (Fig. 2D). Cytoplas- mic staining for melan A was identified in 65% of the cases. Staining with this marker often proved to be patchy and weak (Fig. 2E). Approximately a fourth of the cases showed focal and weak expression of CAM 5.2 (9/40) (Fig. 2F), whereas very rare cases showed weak but dif- fuse positivity for chromogranin A (2/40; 5%) (Fig. 2G) and Pax8 (1/40; 2.5%). None of the cases showed any reactivity with HMWCK. The Ki67 index ranged from <5% to 20% with 13 cases demonstrating a high (> 5%) and 27 cases showing a low (<5%) proliferation rate. Immunohistochemical staining results by tumor subtype are summarized in Table 4.
DISCUSSION
There is a wide variety of neoplasms that can arise in the retroperitoneum. Among these, primary ACC are rare but important tumors to recognize due to their ag- gressive biological behavior. The histologic diagnosis of these tumors is often difficult, and may be further com- plicated by their morphologic heterogeneity. Discrim- ination from other adrenal or extra-adrenal neoplasms may thus prove challenging. In this context, the use of immunohistochemistry as a helpful diagnostic tool be- comes important and over the years, a range of markers have been proposed as an aid in the differential diagnosis of ACC and related tumors. Despite all these efforts, to date no single specific immunohistochemical marker for ACC exists, often making it necessary to use a wider panel of antibodies for confirmation of the diagnosis.
Although prior studies have often relied on investigat- ing small tumor samples arranged in tissue microarrays or
:A
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have applied a limited number of immunohistochemi- cal markers, in the present study we have performed a wide panel of traditional and novel immunohistoche- mical markers on a series of whole section ACC with the aim to comprehensively assess the immunohisto- chemical phenotype of these tumors. In addition, Ki67 was applied to investigate the proliferation index of these lesions.
Among the most important traditional markers that have been studied in adrenocortical neoplasms are in- hibin-a, melan A, calretinin, synaptophysin, chromogra- nin A, and various cytokeratins. Inhibin-a has long been advocated to be useful in the differential diagnosis of adrenocortical neoplasms as it shows consistent im- munoreactivity in normal adrenocortical tissue, adreno- cortical hyperplasia, and adrenocortical neoplasms.5-11 The reported reactivity for ACC has ranged from 68% to 100% and was independent of the functional status of the tumors. Our cases showed largely diffuse cytoplasmic reactivity in 92.5% of cases confirming the usefulness of inhibin-a for the detection of carcinomas of adrenocort- ical origin.
Expression of melan A was initially thought to be restricted to melanocytes and is primarily used in the di-
agnosis of malignant melanoma but it has increasingly been described in a range of steroid-producing tissues and their tumors including ACC.6,8,10,12-15 Immunoreactivity for this marker in adrenocortical tumors is thought to be due to cross reactivity with other epitopes rather than true expression of melan A antigen. In the reported prior series, the largest of which included 40 cases of ACC ar- ranged in a tissue microarray,6 this marker was positive in 55% to 100% of cases. These results are supported by our own finding of 65% positive cases. It has to be noted, however, that staining may not always be diffuse but rather weak and patchy in some cases.
Calretinin is a marker best known for the diagnosis of malignant mesothelioma but has also been demon- strated in steroid-producing tumors including adrenono- cortical neoplasms.11,16 When applied to ACC, calretinin seems to be a consistent marker for this tumor, and re- ported series have been able to show reactivity in 70% to 98.6% of cases. Interestingly, one of the studies reports a pure cytoplasmic staining pattern in ACC contrary to the dual nuclear and cytoplasmic reactivity typically seen in mesothelioma. In our cases, nuclear and cytoplasmic staining was identified in 80% of cases confirming the value of this marker in the context of ACC.
| Subtypes | HMWCK | CAM5.2 | Inhibin | Melan A | Chromogranin | Synaptophysin | Calretinin | SRC-1 | Pax8 | Ki67 (%) |
|---|---|---|---|---|---|---|---|---|---|---|
| Conventional (n = 18) | 0/18 (0) | 3/18 (17) | 16/18 (89) | 10/18 (56) | 1/18 (6) | 12/18 (67) | 13/18 (72 | 17/18 (94 | 1/18 (6) | < 5-20 |
| Oncocytic (n = 17) | 0/17 (0) | 4/17 (24) | 16/17 (94) | 13/17 (76 | 1/17 (6) | 14/17 (82) | 14/17 (82) | 17/17 (100) | 0/17 (0) | < 5-20 |
| Rhabdoid (n = 2) | 0/2 (0) | 2/2 (100) | 2/2 (100) | 1/2 (50) | 0/2 (0) | 1/2 (50) | 2/2 (100) | 2/2 (100) | 0/2 (0) | < 5-10 |
| Myxoid (n = 2) | 0/2 (0) | 0/2 (0) | 2/2 (100) | 2/2 (100) | 0/2 (0) | 2/2 (100) | 2/2 (100) | 2/2 (100) | 0/2 (0) | < 5 |
| Sarcomatoid (n = 1) | 0/1 (0) | 0/1 (0) | 1/1 (100) | 0/1 (0) | 0/1 (0) | 0/1 (0) | 1/1 (100) | 1/1 (100) | 0/1 (0) | 15 |
HMWCK indicates high-molecular weight cytokeratin; SRC-1, steroid receptor cofactor-1.
As one of the biggest issues in the diagnosis of ACC is its separation from another adrenal neoplasm, namely pheochromocytoma, synaptophysin and chromogranin A are of invaluable use when confronted with a biopsy of the adrenal gland. Although these markers typically show strong and diffuse staining in cases of pheochromocytoma highlighting the neuroendocrine nature of these tumors, ACC are usually negative for chromogranin A.7,17 How- ever, we observed chromogranin A staining in 2 of our 40 (5%) cases of ACC, yet the staining was very weak. Al- though concurrent positivity of these 2 cases for melan A, inhibin, or SRC-1 resulted in a pattern favoring a diag- nosis of ACC, these cases highlight the fact that a small subset of ACC may also express chromogranin A. Syn- aptophysin, in contrast, is a marker commonly expressed not only in pheochromocytoma but also in up to 83% of ACC in prior reported series.7,17,18 This was further supported by positive staining of 72.5% of our cases.
A range of antibodies directed against epithelial epitopes have been previously used in cases of ACC. In 1993, Tartour et al17 stained 18 ACC with cytokeratin AE1/AE3 and epithelial membrane antigen (EMA) among other markers, resulting in 36% and 17% of positive cases, respectively. Similar observations were made in later reports stating positivity for pancytokeratin in up to 54.1% and CAM5.2 in up to 10% of cases. Cytokeratins 7 and 20, EMA, and MOC31 were uni- formly negative.6,10,19 We studied 2 epithelial markers, 34BE12 and CAM5.2, covering the high-molecular and low-molecular weight spectrum resulting in 0% and 22.5% of positive cases, respectively.
Steroid factor-1 (SF-1) is a novel transcription factor that plays an important role in the embryologic develop- ment and biological function of steroidogenic tissues in- cluding the adrenal gland.2º Although not an exclusive marker for adrenocortical differentiation, nuclear reactivity for SF-1 antibody is regarded as highly specific and sensi- tive for tumors of adrenocortical origin.7,19,21,22 With pos- itive staining in as much as 89% to 100% of investigated ACC cases, this antibody is currently deemed the best available immunohistochemical marker for adrenocortical differentiation, not only based on its high specificity and sensitivity, but also because of nonreactivity in non- steroidogenic tumors and clean nuclear reactivity devoid of any cytoplasmic background staining.7,19,22 In this study, we used a related marker, SRC-1, a coactivator potentiat- ing the activity of SF-1, and observed diffuse intermediate to strong staining in 39 of 40 cases (97.5%). Our results indicate that this marker has similar staining properties as SF-1, confirming these 2 antibodies as robust markers in the detection of tumors of adrenocortical origin.
Pax8 is a member of the paired box gene family and is a crucial transcription factor for the organogenesis of several organ systems including the thyroid gland, kidney, and Müllerian tract.23-25 Pax8 has been shown to be immunohistochemically expressed in a high percentage of thyroid, renal, thymic, and Müllerian tract neo- plasms.23,26-33 Especially the fact that a wide range of renal tumors shows nuclear reactivity for this marker
make assessment of its expression pattern in ACC very important, yet to date, ACC have only rarely been in- vestigated in this context with negative staining in the reported cases.30-32 In our series of 40 ACC, only a single case showed weak nuclear positivity for Pax8 making this antibody a very useful tool in the differential diagnosis not only with renal tumors but also with other Pax8- positive neoplasms.
In our immunohistochemical study performed on 40 whole tissue sections of 40 ACC, we could largely confirm the results of prior reports often using tissue microarrays as the basis of their investigations. Although still no single marker exists that exclusively labels tumor of adreno- cortical origin, STF-1 or SRC-1 are very useful as markers for tumors of steroid-producing tissues. In- clusion of these antibodies in a panel of markers chosen based on the pertinent differential diagnosis is of utmost value in this context. Likewise, we could demonstrate that Pax8 is very beneficial as a negative marker as expression of this marker almost rules out a diagnosis of ACC. Another observation that can be drawn from the current study is that the immunohistochemical profile seems to be similar in all ACC irrespective of tumor subtype. We did not observe any obvious difference in the staining pattern of different variants; however, it has to be noted that some subtypes were only represented in small numbers, making larger studies evaluating this feature desirable.
Use of the antibodies discussed is particularly ef- fective in the differential diagnosis of the most important tumors that can histologically mimic ACC including renal cell carcinoma, hepatocellular carcinoma, pheochromo- cytoma, and metastatic carcinoma (Table 5). Pax8 is known to be widely expressed in normal and neoplastic renal tissues32 and serves as a useful marker for renal epithelial tumors, including clear cell renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, renal oncocytoma, sarcomatoid renal cell carcinoma, and medullary renal cell carcinoma.32,34 In addition to Pax8, renal epithelial neoplasms are known to express pancytokeratin and CAM5.2, whereas they are generally negative for SF-1, inhibin-a, and melan A.6,22 Apart from CAM5.2, the opposite is true for ACC and inclusion of these markers in a panel is advised in such a scenario. In a similar manner, hepatocellular carcinoma is usually diffusely positive for pancytokeratin and CAM5.2 but negative for SF-1, inhibin-a, and melan A.6,22 The typical immunohistochemical phenotype for pheochro- mocytomas is characterized by positivity for synapto- physin, chromogranin A and negative staining for calretinin, inhibin-a, melan A, and SF-1,11,22,29 separating these tumors from ACC. When a suspicion of metastatic carcinoma exists, the panel to apply will have to include antibodies that are more specific for the suspected origin based on the morphologic and clinical impression, for instance thyroid transcription factor-1 when lung carci- noma is suspected. In general, however, epithelial mark- ers should help in this context, as with the exception of CAM5.2, these should show an opposite staining pattern in ACC versus metastatic carcinoma.
| TABLE 5. Differential Expression of Immunohistochemical Markers in the Differential Diagnosis of Adrenocortical Carcinoma | ||||
|---|---|---|---|---|
| Antibodies | Adrenocortical Carcinoma | Pheochromocytoma | Renal Cell Carcinoma Hepatocellular Carcinoma | Metastatic Carcinoma |
| SRC-1 | + | - | - - | +/- |
| Inhibin | + | - | - | - |
| Calretinin | + | - | - - | - |
| Synaptophysin | + | + | - | |
| Chromogranin A | - | + | - - | - |
| Melan A | + | - | - - | - |
| CAM5.2 | +/- | + + | + | |
| HMWCK | + + | + | ||
| Pax8 | - | - | + - | - |
HMWCK indicates high-molecular weight cytokeratin; SRC-1: steroid receptor cofactor-1.
Lastly, it has to be mentioned that there is currently no reliable marker to distinguish between benign and ma- lignant adrenocortical neoplasms. Using Ki67 and cut-off values of <5% for low proliferative index and ≥ 5% for high proliferative index, Soon et al35 could demonstrate a low proliferation rate in all of their adrenocortical ad- enomas and 30% of ACC, whereas a high labeling index was seen in 70% of ACC. Ki67 was thus thought to be able to differentiate between the 2 tumors. Our results showed that 67.5% of our ACC actually demonstrated a low pro- liferation index suggesting that morphologic criteria, such as the Weiss scoring criteria,4 are still the gold standard and necessary to distinguish between these tumors.
In summary, we have described the immunohisto- chemical phenotype of a large series of full section ACC. Our results largely confirm the findings of previous studies in terms of reactivity for more traditional markers (inhibin- a, melan A, calretinin, synaptophysin, chromogranin, and cytokeratins) as well as SRC-1. In addition, we could clearly demonstrate that Pax8 has value as a negative marker for ACC making this antibody a useful adjunct in the workup of adrenal lesions. A panel of immuno- histochemical markers to include SF-1/SRC-1, inhibin-a, calretinin, and HMWCK in the more general setting or a tailored combination of markers for a more specific differential diagnosis is still needed for the appropriate assessment of any adrenal tumor.
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