CrossMark
Immunohistochemical Biomarkers of Adrenal Cortical Neoplasms
Ozgur Mete 1 . Sylvia L. Asa 1 . Thomas J. Giordano2 . Mauro Papotti3 . Hironobu Sasano4 . Marco Volante5
C Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Careful morphological evaluation forms the basis of the workup of an adrenal cortical neoplasm. However, the adoption of immunohistochemical biomarkers has added tremendous value to enhance diagnostic accuracy. The authors provide a brief review of immunohistochemical biomarkers that have been used in the confirmation of adrenal cortical origin and in the detection of the source of functional adrenal cortical proliferations, as well as diagnostic, predictive, and prognostic biomarkers of adrenal cortical carcinoma. In addition, a brief section on potential novel theranostic biomarkers in the prediction of treatment response to mitotane and other relevant chemotherapeutic agents is also provided. In the era of precision and personalized medical practice, adoption of combined morphology and immunohistochemistry provides a new approach to the diagnostic workup of adrenal cortical neoplasms, reflecting the evolution of clinical responsibility of pathologists.
Keywords Immunohistochemistry · SF-1 . CYP11B2 . CYP11B1 . IGF-2 . Ki67 . p53 . Beta-catenin . Mitotane . Adrenal cortical carcinoma
Introduction
While careful morphological evaluation is the foundation of the workup of an adrenal neoplasm, the adoption of immunohisto- chemical biomarkers adds value at many levels to a diagnostic evaluation. This “modern” approach of combining morphology and ancillary immunohistochemical biomarkers has also im- proved the clinical management of patients with adrenal cortical carcinomas. In this review, the authors provide a brief summary of immunohistochemical biomarkers that have been used in the confirmation of adrenal cortical origin and differentiation that allows functional correlations of adrenal cortical proliferations
☒ Ozgur Mete ozgur.mete2@uhn.ca
1 Department of Pathology, University Health Network, 200 Elizabeth Street, 11th floor, Toronto, ON M5G 2C4, Canada
2 Departments of Pathology and Internal Medicine, University of Michigan Health System, Ann Arbor, MI, USA
3 Department of Pathology, Turin University at Molinette Hospital, Turin, Italy
4 Department of Pathology, Tohoku University School of Medicine, Sendai, Japan
5 Department of Oncology, University of Turin at San Luigi Hospital, Turin University, Orbassano, Turin, Italy
as well as biomarkers of diagnostic, predictive, and prognostic significance in patients with adrenal cortical carcinoma. In ad- dition, a brief section on potential additional theranostic bio- markers in the prediction of treatment response to mitotane and other relevant chemotherapeutic agents is also provided.
Confirmation of Adrenal Cortical Origin
The confirmation of adrenal cortical origin is the first step that a diagnostician should undertake when dealing with an adre- nal mass [1]. A recent paper underscored a major diagnostic pitfall in the workup of adrenal masses which was linked to the misinterpretation of adrenal cortical lesions either as med- ullary or non-adrenal origin [2]. The use of relevant bio- markers in the distinction of primary adrenal cortical origin of a non-functional neoplasm is highly recommended in both surgical resections and small biopsy specimens (e.g., fine nee- dle aspiration biopsy and core biopsy) [3].
Several biomarkers have been proposed to confirm adreno- cortical origin (Table 1, Fig. 1). However, one should recog- nize the specificity and sensitivity of various biomarkers, as the adrenal gland and retroperitoneum host various neoplasms that can pose diagnostic challenges during the preoperative workup. Steroidogenic factor 1 (SF-1) is considered the most reliable and specific, since this nuclear transcription factor (Fig. 1a) is expressed in nontumorous adrenal cortex as well
| Biomarker | ACC | PHEO/PGL | RCC | Metastatic carcinoma |
|---|---|---|---|---|
| Pan-cytokeratin | -/(+) | – | + | + |
| Vimentin | + | -/(+) | + | -/+ |
| Chromogranin-A | – | + | – | - (+ in NE carcinomas) |
| Synaptophysin | + | + | – | - (+ in NE carcinomas) |
| Melan A | + | – | (+ in some) - | _a |
| Calretinin | +/- | – | – | -/+ |
| Alpha-inhibin | +/- | -/(+) | – | -/+ |
| D2-40 | + | – | – | -/+ |
| Tyrosine hydroxylase | – | + | – | – |
| CD10 | +/- | NA | +/- | -/+ |
| SF-1 | + | - | - | – |
| PAX8 (monoclonal) | – | – | + | +/- |
ACC adrenal cortical carcinoma, PHEO pheochromocytoma, PGL paraganglioma, RCC renal cell carcinoma, NE neuroendocrine, NA not available or limited data
ª This biomarker is also expressed in several other non-epithelial neoplasms including melanoma and PEComas
as in adrenal cortical neoplasms [1]. SF-1 is indeed the hall- mark of steroidogenic tissues; it is expressed in the steroido- genic cellular components and tumors arising from adrenals and gonads as well as in the gonadotrophs of the pituitary gland and functions as a master regulator of steroidogenesis [4, 5]. In a large series of adrenal cortical and non-cortical neoplasms, the overall specificity of SF-1 was reported to be
100%, and its sensitivity was reported as 100 and 98% in benign adrenal cortical proliferations (adrenal cortical hyper- plasia and adenoma) and adrenal cortical carcinomas, respec- tively [6]. While various commercially available antisera pro- vided comparable results, monoclonal antibodies against SF-1 were proven to be more accurate [7]. An additional value of SF-1 is its potential role as a prognostic marker in adrenal
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cortical carcinoma as higher SF-1 mRNA and protein expres- sion is associated with poor prognosis [3, 7].
Another widely used biomarker is Melan A (also known as MART-1) (Fig. 1b) which has been well-described in adrenal cortical as well as in other steroidogenic tissues, nearly 20 years ago [8]. The specificity and sensitivity rates of Melan A immunohistochemistry are very high, provided that a diagnosis of metastatic melanoma or primary adrenal PEComa (e.g., epithelioid angiomyolipoma) is excluded with the use of other melanocyte-specific or myoid markers, re- spectively [9].
Other biomarkers of adrenal cortex share positive immuno- reactivity with other steroidogenic tissues, but their overall specificity and sensitivity-especially if used alone-are thought to be lower than those of SF-1 and Melan-A, and their pattern of positivity might be variable, if not only focal. These biomarkers include alpha-inhibin (Fig. 1c), calretinin (Fig. 1d), and D2-40 [1]; of note, both D2-40 and calretinin can also be expressed in mesothelial cells and related neoplasms that can also manifest in the adrenal gland [1, 10].
In the setting of special adrenal cortical carcinoma var- iants, i.e., oncocytic and myxoid, most adrenocortical tissue-specific markers including SF-1 are generally pre- served, although some peculiarities were reported, such as expression of neurofilaments in the myxoid type of ad- renal cortical carcinoma [11]. A rare adrenocortical carci- noma variant that can pose diagnostic challenge is the sarcomatoid variant of adrenal cortical carcinoma, in which adrenal cortical-specific tissue biomarkers are either negative or restricted to residual epithelioid/differentiated components [12]. These observations were confirmed by a recent genomic study that included two sarcomatoid adre- nal cortical carcinomas [13]. Instead, the sarcomatoid var- iant of adrenal cortical carcinoma can express markers of epithelial-to-mesenchymal transition that are of poor diag- nostic utility in routine diagnostic workup [12].
The rational of the selection of biomarkers should be spe- cifically designed according to the clinical setting, the ob- served morphological pattern, and the laboratory resources. A minimal dataset for the most frequent situations is briefly proposed and discussed here. The distinction of adrenal med- ullary from cortical differentiation is quite simple and relies on a limited panel of markers [2] including chromogranin-A and/ or tyrosine hydroxylase (the rate limiting enzyme in catechol- amine synthesis), SF-1, and/or Melan A (if SF-1 is not avail- able); this is especially important for tumors displaying oncocytic or clear cell cytomorphology or myxoid features. It is important to note that synaptophysin is not a reliable biomarker of adrenal medulla or neuroendocrine differentia- tion since it can also be expressed in adrenal cortical cells and their proliferations [14]. Rather, synaptophysin expression in the setting of negative chromogranin supports cortical differentiation.
Adrenocortical neoplasms with clear cells-mostly adrenal cortical adenomas-might need to be distinguished in some situations from clear cell-renal cell carcinoma. In most situa- tions, the morphology and clinical grounds alone are adequate for a correct diagnostic interpretation. However, the situation might be a bit more complex in the case of eosinophilic tumors that might be ascribable to both adrenocortical neoplasms and renal cell carcinomas other than clear cell variant. Today, the gold standard requires the identification of nuclear PAX8 for renal cell carcinoma. Prior to the use of the monoclonal PAX8 antibody in the diagnosis of renal cell carcinoma, CD10 was used for this distinction; however, CD10 is expressed in adre- nal cortical carcinomas as well as in other neoplasms [15]. In this context, positivity for SF-1, Melan A, calretinin, alpha- inhibin, and D2-40 are more sensitive and specific markers of adrenocortical origin [16]. Nevertheless, one should also rec- ognize that a subset of renal cell carcinomas can also express Melan-A [17].
Another frequent diagnostic situation is the differential diagnosis between adrenal cortical neoplasm and metasta- ses, with special reference to those of epithelial origin. In such situations, one should always remember the limita- tions of biomarkers and consider using more than a single stain for confirmation. While the relevant clinical history might not be available or may be unknown at the time of diagnostic workup, staining for SF-1 and Melan-A is ad- vised as well as markers of potential primary lesions. In our experience, staining for pan-keratin, cytokeratins 7 and 20, EMA, and site-specific transcription factors (e.g., TTF-1, CDX-2) as well as SF-1 and Melan A are very helpful in the distinction of metastatic carcinoma from an adrenal cortical neoplasm.
Finally, primary mesenchymal adrenal tumors, such as sar- comas with epithelioid morphology, might pose diagnostic problems in their distinction from adrenal cortical prolifera- tions, again mostly carcinomas. Among them, angiosarcoma is indeed the most frequent in this setting (although very rare in terms of overall prevalence); pan-cytokeratin can show fo- cal patterns similar to adrenal cortical carcinoma, but lineage- specific markers (SF-1 and Melan A on one side and endothelial markers on the other side) can easily solve the problem when the correct differential diagnosis is considered, although exceptional cases showing a combination of angiosarcoma with adrenal cortical neoplasms might be more complex to define [18, 19].
The distinction between a sarcoma and sarcomatoid variant of adrenal cortical carcinoma is often a real challenge since the latter can be negative for SF-1. In this context, the distinction of SF-1-negative and mdm2-positive sarcomatoid neoplasm in the adrenal gland should not be considered as an unequiv- ocal evidence of dedifferentiated liposarcoma as mdm2 can also be expressed in several neoplasms including adrenal cor- tical carcinomas [20].
Definition of Functional Differentiation in Adrenocortical Cells
The ultrastructural features of an adrenal cortical neoplasm can be used in the distinction of an aldosterone-producing adrenal cortical neoplasm from other adrenal cortical neoplasms (e.g., glucocorticoid-secreting and non-functional adrenal cortical neo- plasms). A simple and efficient morphological evaluation based on the demonstration of atrophy of the nontumorous adrenal cortex confirms the presence of a glucocorticoid-secreting adre- nal cortical proliferation in the absence of exogenous cortisol administration [21, 22]. While the use of adrenal vein sampling facilitates the distinction of bilateral adrenal cortical proliferations leading to primary aldosteronism, the identification of spironolactone bodies in an adrenal cortical neoplasm and adja- cent paradoxical zona glomerulosa hyperplasia often confirms the source of primary aldosteronism in the setting of spironolactone treatment [23, 24].
Primary aldosteronism accounts for around 5-10% of pa- tients with hypertension and is the leading cause of secondary hypertension and endocrine hypertension [25-27]. The chal- lenge in the distinction of the source of primary aldosteronism in surgical specimens has been well known. Not all patients are treated with spironolactone, and new medications used in the setting of primary aldosteronism disable the ability to de- tect treatment-related change in functional cells. In addition, it has been shown that radiologically identified adrenal cortical nodules may not necessarily be the source of primary aldoste- ronism [28]. Moreover, patients may present with multiple cortical proliferations, and often, one of the cortical nodular proliferations may show somatic mutation [29]. In fact, some of these are related to clinical, morphological, and steroido- genic heterogeneity that are also reflected in the molecular characteristics of adrenal cortical neoplasms [21, 23, 24]. Therefore, the accurate distinction of functional differentiation may be difficult based only on the cytomorphological details of an adrenal cortical proliferation [30, 31].
Several biomarkers have been suggested in the assessment of functional differentiation in adrenal cortical proliferations leading to primary aldosteronism [32]. Among these, mono- clonal antibodies against steroidogenic enzymes, especially those against HSD3B1/2 (3ß-hydroxysteroid dehydrogenase type 1 and type 2) and CYP11B1/2 (Cytochrome P450 family 11 subfamily B member 1 and member 2), have garnered significant interest [33, 34]. These monoclonal antibodies were not only shown to distinguish these isoforms from each other, but more importantly, these biomarkers have been shown to enable diagnosticians to visualize the cellular prolif- eration responsible for the synthesis of corresponding steroid hormones (e.g., aldosterone and cortisol) in surgical speci- mens [33-36] (Fig. 2). In addition, the application of these biomarkers has helped to develop genotype-phenotype corre- lations in the many faces of primary aldosteronism [24].
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Yamazaki et al. demonstrated that using the monoclonal CYP11B2 (aldosterone synthase) antibody, CYP11B2- expressing adrenal cortical proliferations which were not iden- tified on cross-sectional images from 25 cases (18 with bilat- eral source of aldosterone production, 7 with unilateral source based on adrenal vein sampling) were histologically classified into multiple adrenal cortical micronodules (MN) and diffuse hyperplasia of the zona glomerulosa layer (DH) based on the
reactivity pattern of CYP11B2-positive cells [37] (Fig. 3). In addition, HSD3B1 and HSD3B2, which also play pivotal roles in the process of aldosterone biosynthesis, were shown to be useful in such presentations [37, 38]. Yamazaki et al. reported abundant HSD3B1/2 immunoreactivity in the non- nodular part of the zona glomerulosa (ZG), as well as in MN when comparing to DH [37] (Fig. 4). In addition, HSD3B2 immunoreactivity was more abundant than HSD3B1 in aldosterone-producing adenomas, but the status of HSD3B1 immunoreactivity in the tumor was significantly correlated with that of CYP11B2 [38] (Fig. 5). The findings of this series along with other observations [31] in the field of primary aldosteronism suggest that the application of specific bio- markers against steroidogenic enzymes is useful in the classi- fication of various histological presentations.
Diagnostic, Predictive, and Prognostic Biomarkers of Adrenal Cortical Carcinoma
Traditionally, pathologists have used morphological findings to distinguish adrenal cortical carcinomas from adenomas. Among these, proliferation rate (often mitotic count), tumor necrosis, and invasive growth (especially vascular invasion) are still regarded as the most critical elements along with the tumor growth pattern. While the diagnosis of invasive adrenal cortical carcinomas with high proliferative features does not pose a diagnostic challenge, adrenal cortical neoplasms with
borderline/atypical features that are confined to the adrenal gland can prove to be difficult. Several histomorphology- based scoring schemes have been developed to tackle these problems [39-46]. Among these, the Weiss scoring scheme has been the most widely accepted algorithm in many prac- tices. In addition to well-documented interobserver variation in the application of the Weiss criteria, this system has not proven useful in all adrenal cortical neoplasms. For instance, it has been shown that myxoid adrenal cortical neoplasms can have unpredictable biology [11]. In addition, oncocytic adre- nal cortical neoplasms require the application of the Lin- Weiss-Bisceglia scheme [46]. More recently, proposed scor- ing schemes, including the Helsinki scoring scheme and the reticulin algorithm, have now been recognized officially in the 4th edition of the WHO classification of adrenal cortical neo- plasms [47]. Unlike the onerous multi-parameter scoring schemes that are subject to interobserver variability, the reticulin algorithm has gained popularity for its simplicity and reproducibility of the demonstration of altered reticulin framework in association of one of the following parameters: vascular invasion, necrosis, and mitotic activity (>5 per 50 high power fields) [41, 42, 48, 49]. Nevertheless, there are adrenal cortical neoplasms that do not readily fit algorithms and remain classified as of uncertain malignant potential [50-52]. This is particularly applicable to fragmented or morcellated laparoscopic adrenalectomy specimens in which the status of invasive growth cannot be assessed, and intra- tumoral proliferative heterogeneity and morphological
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heterogeneity (e.g., adenoma-like areas in adrenal cortical car- cinoma) that may reflect an adenoma-carcinoma progression sequence in some neoplasms [1, 48, 50, 53-55]. Increasing demands for accurate diagnosis on smaller biopsy specimens also challenge diagnosticians. A high-grade adrenal cortical carcinoma with altered reticulin framework and increased mi- totic activity can be distinguished in a core biopsy; however, given the heterogeneity in these neoplasms, the distinction of a non-metastatic low-grade adrenal cortical carcinoma and adenoma can be a real challenge based on morphological evaluation alone.
Advances in our understanding of the molecular biology of adrenal cortical carcinomas showed that the transcriptome profiling and 68-CpG probe DNA-methylation signature of an adrenal cortical neoplasm can be used to distinguish ma- lignancy and can also provide prognostic information [13, 56, 57]. Despite this significant progress, most practices still fail to integrate those tests as a part of the routine practice due to cost and barriers to access. Therefore, attempts to validate translational biomarkers have proven of value using immuno- histochemistry [48, 58-69]. Among these, IGF-2 immunohis- tochemistry has been shown to be the best translational diag- nostic biomarker of adrenal cortical carcinoma irrespective of cytomorphology and proliferative grade of the tumor [48]. In fact, the diagnostic utility of this biomarker is not surprising as despite genomic and biological differences among various molecular clusters of adrenal cortical carcinoma, IGF-2
overexpression was consistently identified in around 90% of adrenal cortical carcinomas irrespective to prognostic molec- ular clustering of these neoplasms [13, 56, 57, 70, 71]. While earlier studies reported variable IGF-2 reactivity in some ad- renal cortical adenomas [58, 60], a recent investigation dem- onstrated that the diagnostic utility of IGF-2 antibody was related to the rigid optimization of the commercially available IGF-2 antibody by subtracting the basal IGF-2 reactivity to reveal a paranuclear (juxtanuclear) reactivity pattern that was unique to adrenal cortical carcinoma [48] (Fig. 6). The juxtanuclear IGF-2 immunoreactivity pattern was linked to altered cellular IGF-2 processing that leads to the accumula- tion of larger precursor forms of IGF-2 in the Golgi apparatus of tumor cells [48, 59, 72, 73].
Another translation biomarker of adrenal cortical carcino- ma is p53. The identification of p53 overexpression has been widely used to support the diagnosis of adrenal cortical carci- noma [1, 61-64] (Fig. 7). However, not all adrenal cortical carcinomas have p53 overexpression and some tumors with p53 alterations can also show global loss of expression. Therefore, one should recognize the limitations of the use of p53 alone in the distinction of adrenal cortical carcinoma. Despite the importance of an altered p53 genotype in adrenal cortical carcinomas, especially poor-prognosis ACCs [13], several cohorts did not find any statistical difference between prognosis and p53 immunohistochemistry [48, 62, 63, 65, 66]. Unlike adults, around 70% of pediatric adrenal cortical
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neoplasms were associated with germline p53 mutations [74]. Pediatric adrenal cortical carcinomas with germline p53 and somatic ATRX mutations were shown to have poor prognosis, larger tumor size, and advanced stage when compared with those harboring germline p53 mutations and wild-type somat- ic ATRX, as well as those with wild-type germline p53 and wild-type somatic ATRX status [74]. Therefore, an immunopanel of ATRX and p53 may be of value in the mo- lecular histopathological prognostication of pediatric adrenal cortical carcinomas.
Another important translation biomarker is beta-catenin immunohistochemistry (Fig. 8). While CTTNB1 alterations and wnt-pathway activation are not restricted to adrenal corti- cal carcinomas [75, 76], molecular profiling of adrenal cortical carcinomas identified CTTNB1 mutations in a subset of adre- nal cortical carcinoma with adverse prognosis [56, 77]. Variable or focal nuclear and cytoplasmic beta-catenin expres- sion is seen in some adrenal cortical carcinomas; however, diffuse nuclear and cytoplasmic immunoreactivity is a feature of adrenal cortical carcinoma, within the appropriate morpho- logical context [48], and typically reflects adverse prognostic molecular clusters [56].
Several other biomarkers have been proposed to facilitate the distinction of malignancy in adrenal cortical proliferations;
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most of these were based on their roles in cell cycle regulation or cellular proliferation including but not limited to Ki67, phosphoHistone-H3, BUB1B, HURP, NEK2, and p27 [48, 59, 66-69, 78]. Among these, Ki67 has been the most popular biomarker as it is widely used in routine diagnostic surgical pathology. There is a general thought that most adrenal corti- cal carcinomas often have a Ki67 labeling index that exceeds 5% and most adenomas display a Ki67 labeling index less than 5% [1, 50, 59, 67, 68]. However, not all adrenal cortical carcinomas show high Ki67 labeling index and some benign adrenal cortical proliferations might have a Ki67 index around this cut-off. PhosphoHistone-H3 immunohistochemistry is al- so helpful to assist a formal mitotic count [78]. In adrenal cortical neoplasms, this approach was shown to provide a better interobserver correlation [78]. More importantly, it can be used to assist the reticulin algorithm as well as the tumor proliferative grade (high-grade adrenal cortical carcinoma: mi- totic activity > 20 per 50 high power fields; low-grade adrenal cortical carcinoma: mitotic activity ≤20 per 50 high power fields) [1, 48, 79, 80]. As with other nuclear antigens (e.g., estrogen receptor), one should be aware that suboptimal tissue fixation may hamper the detection of nuclear
immunoreactivity. This is also applicable to other nuclear bio- markers used in the assessment of adrenal cortical neoplasms such as SF-1 and Ki67 (unpublished observation of Drs. Mete and Asa) and tumor heterogeneity that is common in adrenal cortical neoplasms makes it important to consider performing Ki67 and phosphoHistone-H3 immunohistochemistry on multiple tumor blocks, especially those with high tumor cel- lularity and mitotic activity. The Ki67 proliferation index, mi- totic tumor grade, and/or other adverse pathological features (e.g., vascular invasion) are typically used in risk stratification as well as to determine the need for adjuvant mitotane treat- ment [1, 50, 80-83]. Therefore, these quantifications should not be performed using estimation [84]; like neuroendocrine neoplasms, manual counting of 1000 tumor cells on color- printed captured images from hot spots or assessment using automated image analysis, nuclear algorithms are the pre- ferred methodologies [85] (Fig. 9).
Immunohistochemistry for BUB1B (a component of the spindle assembly checkpoint regulating chromosome segrega- tion during mitosis [86], HURP (kinetochore protein main- taining mitotic spindle integrity) [87, 88], and NEK2 (part of kinases involved in the spindle assembly and centrosome cy- cle) [89, 90] was also shown to be useful in the distinction of adrenal cortical carcinomas [48]. The use of these three bio- markers is currently not the standard of practice due to their lesser extent of expression (especially HURP and NEK2) as well as lack of access for routine clinical use. However, at a molecular level, BUB1B overexpression was identified in a subset of adrenal cortical carcinomas in the poor prognostic group [56]. While global experience on BUB1B immunohis- tochemistry is currently lacking, a recent series reported en- richment of BUB1B immunoreactivity in high-grade adrenal cortical carcinoma [48].
Recognition of genetic susceptibility related to germline ge- netic defects is increasingly important, especially in endocrine pathology practice. While clinical history and the assessment of the nontumorous gland, for example the presence of features of Primary Pigmented Nodular Adrenal Disease (PPNAD), may help in some cases, the application of immunohistochemical bio- markers such as beta-catenin and APC (FAP syndrome), p53 (Li- Fraumeni syndrome), menin (MEN1 syndrome), and p27 (MEN4 syndrome) can assist diagnosticians [1, 54, 91]. IGF-2 immunohistochemistry in the distinction of Beckwith- Wiedemann syndrome may be of limited value in adrenal cortical carcinomas arising in this background. Recently, extra-colonic manifestations of Lynch syndrome (Fig. 10) and SDH-related familial paraganglioma syndrome have expanded the genetic susceptibility to adrenal cortical neoplasia [92, 93]; therefore, MMR and SDHB immunohistochemistry are also useful. While some experts routinely perform a panel approach, espe- cially in young patients without any clinical history, the use of MMR immunohistochemistry is advised by others in all seem- ingly sporadic adrenal cortical carcinomas [1].
Endocr Pathol
Layer Attributes
Percent Positive Nuclei
51.4079
Intensity Score
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[3+] Percent Nuclei
39.7426
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[2+] Percent Nuclei
5.39019
[1+] Percent Nuclei
6.27514
[0+] Percent Nuclei
48.5921
Average Positive Intensity
134.969
Average Negative Intensity
236.469
[3+] Nuclei
988
[2+] Nuclei
134
[1+] Nuclei
156
[0+] Nuclei
1208
Total Nuclei
2486
Average Nuclear RGB Intensity
134.751
Average Nuclear Size (Pixels)
228.605
Average Nuclear Size lum
57.0828
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4-XO.
Region
Length (um) |Area [um2]
Text
Percent Positive Nuclei
1 ☒
1
2938
401892
51.4079
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Prediction of Response to Mitotane Therapy and Other Potential Theranostic Biomarkers in Adrenal Cortical Carcinoma
Mitotane, which is considered to have an adrenolytic effect, is the most commonly used drug for the treatment of advanced adrenal cortical carcinoma in combination with chemothera- peutic regimens that often contain platinum derivatives, etoposide, doxorubicin, and adriamycin, as in the EDP-M scheme [94]. Mitotane alone is also used in the adjuvant set- ting [95]. On the other hand, the role of radiotherapy is very limited and considered in select situations.
Currently, the clinical, histological, immunohistochemical, and molecular biomarkers that predict response to these treat- ment options and/or explain mechanisms of resistance or pro- gression are largely missing in the field of adrenal cortical carcinoma. Therefore, there are no well-established surrogate biomarkers to determine response to clinically validated treat- ment modalities [82]. One of the most relevant reasons is that the anti-neoplastic effects of mitotane are unclear. Mitotane is active through its metabolization into two substances: o,p’- DDA and o,p’-DDE. The mechanisms which have been hy- pothesized to explain both anti-neoplastic properties and side effects include its interaction with cellular membranes [96] that also regulate its ligation with serum lipoproteins and availability [97]. Moreover, mitotane activation has been linked to the expression of members of the P450 cytochrome
automated image analysis nuclear algorithms are the preferred methodol- ogies. This photomicrograph illustrates the assessment of the MIB-1 pro- liferation index in a metastatic adrenal cortical carcinoma in liver. The assessment was performed using an automated image analysis nuclear algorithm
family. Ronchi et al. reported differential CYP2W1 (one of the P450 cytochrome family members) mRNA expression pro- files in non-adrenal tissues and those of normal and neoplastic adrenals [98]. The same study demonstrated that higher CYP2W1 immunoreactivity was associated with improved response to mitotane therapy, either in the setting of adjuvant or palliative care of adrenal cortical carcinoma [98]. While one can hypothesize that the specific activity might be related to the oxidation of mitotane by CYP2W1, functional studies using adrenal cortical cell lines are still missing to establish the role of CYP2W1 in the cytotoxic effects of mitotane.
Another molecule which has been linked to mitotane efficacy is the ribonucleotide reductase M1 (RRM1) [99]. Low RRM1 immunoreactivity and gene expression were reported to predict an increased sensitivity to mitotane in a cohort of adrenal cortical carcinoma patients treated with adjuvant mitotane protocols [99]. Consistent with this observation, in patients with low RRM1 gene expression, adjuvant mitotane was associated with im- proved disease-free survival [99]. However, the mechanisms of this association are largely unknown, since the RRM1 is an en- zyme involved in the synthesis of deoxyribonucleotides for DNA synthesis and represents the cellular target for gemcitabine [100]. In a subsequent functional study, it has been shown that RRM1 probably is not a direct target of mitotane but exerts an indirect effect, interfering, when expressed at high levels, with the intra- cellular transformation of mitotane into o,p’DDE and o,p’DDA [101]. However, the role of RRM1 immunohistochemistry in the
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prediction of response to mitotane still requires further clinical validation.
As concerns relevant biomarkers linked to chemotherapy responsiveness, excision repair cross complementing group 1 (ERCC1) plays a critical role in the repair of platinum-induced DNA damage. ERCC1 has been proposed as a marker predic- tive of response to platinum-based regimens in several cancer types, especially in non-small cell lung cancer. Indeed, the major problem is related to the poor methodological reproduc- ibility of ERCC1 [102], as several antibodies that have been developed and proposed have had inconsistent results. In fact, few studies investigating the role of ERCC1 immunostaining as a predictive marker for the response to platinum-based che- motherapy in adrenal cortical carcinoma had reported conflict- ing results, although recently a positive correlation in a large series was reported [103].
Topoisomerase II alpha (TOP2A) is a well-known prognostic parameter in several neoplasms and predicts the efficacy of
anthracyclines. A recent investigation showed that high TOP2A gene expression (and to a lower extent protein expression) is not prognostic in patients with adrenal cortical carcinoma, but it is associated with longer progression-free time in those exposed to the EDP-M protocol [104]. As for gemcitabine-containing regi- mens, which are a second line chemotherapeutic intervention, RRM1 has been recently investigated in adrenal cortical carcino- ma but failed to have any predictive value [105].
Among new potential biomarkers that appeared recently on the horizon of potential theranostic biomarkers in adrenal cor- tical carcinoma, some interest has been dedicated to VAV2. This guanine nucleotide exchange factor for small GTPases is directly regulated by SF-1, promotes cell invasion in adrenal cortical cancer [106], and is associated with poor prognosis in patients with this disease [107]. The recent development of inhibitors of RAC1-VAV2 association has provided proof-of- principle that this interaction represents a druggable target in adrenal cortical carcinoma [108].
Conclusion
This review provides a brief overview of immunohistochem- ical biomarkers of adrenal cortical neoplasms. In the era of precision and personalized medical practice, adoption of com- bined morphology and immunohistochemistry represents an improved approach to the diagnostic workup of adrenal corti- cal neoplasms. While some of the biomarkers discussed are currently used for research only, the majority have practical application that enhances the clinical importance and contri- bution of pathologists and the care of patients.
Acknowledgements The authors would like to thank Dr. Celso E. Gomez-Sanchez for providing the CYP11B1/2 monoclonal antibody for immunohistochemistry and Dr. Masao Doi and Dr. Hitoshi Okamura for providing the HSD3B1/2 monoclonal antibody.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflicts of interest.
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