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Expert Opinion
1. Introduction
2. Macroscopic findings pertinent to differential diagnosis
3. Histological differentiation diagnosis between adrenocortical adenoma and carcinoma
4. Molecular and cellular diagnosis of adrenocortical carcinoma in resected adrenal tumors
5. Conclusion
6. Expert opinion
informa healthcare
Discerning malignancy in resected adrenocotical tumors
Sasano Hironobu+, Suzuki Takashi & Nakamura Yasuhiro
* Tohoku University School of Medicine, Department of Pathology, 2-1 Seiryou-machi, Aoba-ku, Sendai, 980-8575, Japan
Background: The number of adrenocortical tumors discovered incidentally has recently increased owing to the advent of imaging techniques. The most important diagnostic point in evaluation of resected specimens of these tumors is to discern malignancy to determine postoperative manage- ment of the patients. Objective/methods: To determine what the effective methods of discerning malignancy in resected specimens of adrenocortical tumors are at this juncture. To provide relevant and practical information pertinent to those involved in the management of patients with adreno- cortical tumors. Conclusion: Careful macroscopic evaluation, including the selection of the specimens submitted and application of the criteria of Weiss for histological diagnosis, is still considered the ‘gold standard’ for diagnosis of adrenocortical carcinoma. However, it is important to recognize its limitations in the diagnosis of adrenocortical oncocytoma and pediatric adrenocortical tumors. The auxiliary methods that may be of clinical relevance at this juncture include the analysis of the Ki67/MIB-1 labeling index and IGF-II expression in the tumors.
Expert Opin. Med. Diagn. (2008) 2(10):1095-1105
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1. Introduction
Adrenocortical carcinoma (ACC) is a relatively rare tumor with a reported annual incidence of 0.5 - 2 cases per million [1]. The great majority of patients with ACC present symptoms related to its endocrine activity, but an increasing number of the patients has recently been detected in those without particular clinical endocrine abnormalities. This is because of the advance of radiological diagnoses such as MRI and CT scan, which can detect an adrenal mass at the time of evaluating symptoms or clinical signs unrelated to adrenocortical disease [2,3].
Nearly a third to a half of the adrenocortical carcinomas are highly malignant tumors. Therefore, when the patients with adrenocortical mass or with adreno- cortical dysfunctions are clinically detected, the first and the most important clinical aspects in the management of these patients are whether that adreanal mass represents malignant neoplasm, or whether or not adrenocortical dysfunction is caused by adrenocortical carcinoma. However, some adrenocortical carcinomas are associated with clinical and/or radiological diagnostic difficulties, despite their recent advances. Therefore, pathologic evaluation of the resected adrenal mass with respect to discerning malignancy plays a very important role in further management of the patients [4]. However, this differential histopathological diagnosis has also been well known to be associated with various diagnostic difficulties. Therefore, in this review, gross and histopathological findings of adrenocortical carcinoma pertinent to the differential diagnosis as well as recent advances in cellular and molecular findings of adrenocortical neoplasms, which may contribute to differential diagnosis of adrenocortical malignancy in resected specimens, will be briefly summarized.
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Discerning malignancy in resected adrenocotical tumors
2. Macroscopic findings pertinent to differential diagnosis
As in disorders arising in other organs, macroscopic or gross evaluation of the surgically resected specimens, which determines the portion submitted to diagnostic histopathology or molecular pathology laboratories, is one of the most important steps in this diagnostic pathway in the management of adrenocortical carcinoma.
The first important step is to obtain the weight of the resected neoplasm as accurately as possible in addition to obtaining its dimensions. The measurement and record of tumor weight still play important roles in the process of evaluation. Although controversial, Tang and Gray reported that all cortical tumors weighing > 95 g were malignant, whereas tumors < 50 g in weight were benign (the average weight of the tumor is 705 g, ranging from 96 to 2460 g) [5]. Van Slooten et al. reported that only tumors with weight > 150 g metastasized in their series [6]. However, small adrenocortical tumors do metastasize and some large tumors do not. The tumor reported by Gandour and Grizzle weighed only 40 g, and its greatest dimension measured only 4 cm, but metastasized 3 years following bilateral adrenalectomy [7]. On the other hand, Hough et al. reported that the tumor weighing 1800 g did not metastasize [8]. Therefore, the weight of the tumor is important in the process of discerning malignancy of resected adrenocortical neoplasms, and the accurate weight of the specimens should be recorded in the histopathology report. However, it is also important to note that the weight itself is not a reliable diagnostic indicator of the resected adrenocortical tumor.
The next important step is careful observation of the gross features of the cut surface of the tumor. Hemorrhage and necrosis on the cut surface are rarely observed in adrenocortical adenoma. Necrosis is sometimes associated with cystic degeneration of the tumors. The presence of necrosis and hemorrhage, therefore, suggests the diagnosis of adrenocortical carcinoma in the resected adrenocortical tumors. However, it is also true that many adrenocortical carcinomas are not associated with foci of necrosis and hemorrhage. In addition, it is also important to sample the specimens from the areas adjacent to the foci of necrosis and hemorrhage when grossing the specimens at diagnostic pathology laboratories. Scientific rationale is behind this sampling process in adrenocortical tumors because the areas adjacent to foci of necrosis or hemorrhage are usually associated with increased cell proliferation or turnover of the tumor cells, which requires abundant blood supply through neovascularization. However, when the rate of tumor cell proliferation exceeds that of tumor stromal cells, especially endothelial cells of neoplastic tissues, subsequent lack of nutritional supply results in necrosis of carcinoma cells, and the immature nature of neovascularization results in permeation of blood components, that is, intratumoral hemorrhage. Therefore, sampling of areas adjacent to the
foci of hemorrhage and/or coagulative necrosis provides the areas that are associated with one of the highest cell turnovers in the whole tumor for subsequent histopathlogical evaluation. However, it is also important to note that foci of intratumoral fibrosis and myxomatous degeneration can be seen in both adenoma and carcinoma [9].
Some reports in the past emphasized the color of the cut surface of the tumor, but that for viable parts of adrenocortical neoplasms is by no means a reliable indicator of adrenocortical carcinoma. Carcinoma may be tan, yellow, or yellow orange on the cut surface. However, it is worthwhile to remember that a homogeneous black cut surface, as observed in black- pigmented adenoma, is rarely present in adrenocortical carcinoma. In addition, a homogenous golden yellow cut surface such as seen in aldosteronoma or adrenocoritical adenoma, associated with primary aldosteronism, has also rarely been reported in adrenocortical carcinomas. Therefore, the color of the cut surface should also be recorded in the histopathological report.
3. Histological differentiation diagnosis between adrenocortical adenoma and carcinoma
The distinction of this so-called ‘well-differentiated’ adrenocortical carcinoma from adenoma may be one of the most diagnostic difficulties in surgical pathology practice. There is no single histological criterion that can reliably differentiate adrenocortical carcinoma from adenoma-like capsular or vascular invasion of thyroid follicular carcinoma. Only the systems that evaluated multiple histological and/or non-histological criteria of the resected cases can provide reliable histological diagnosis. Three different histological scoring systems have been proposed by various investigators, and all systems are equally useful for predicting clinical outcome of the patients with resected adrenocortical neoplasms [6,8,10,11]. However, among these three criteria, the criterion of Weiss has been most widely used, and it has become the ‘gold standard’ of histological diagnosis of adrenocortical carcinoma, as seen in its incorporation into the recent World Health Organization tumor classification [12]. Therefore, in this review, we shall review this histological criterion of Weiss from the standpoints of advantages and limitations in terms of managing the patients with adrenocortical neoplasms.
In 1984, Weiss proposed nine histological criteria (Box 1), which is important in evaluating adrenocortical malignancy based on extensive retrospective clinicopathological studies of the cases of adrenocortical neoplasms with known clinical outcome, especially which histological factors influence the clinical outcome of the patients with the tumor [10]. Weiss subsequently lowered the threshold for adreno- cortical malignancy from 4 to 3 histologic criteria because 20 of 23 cases that fulfilled 3 histologic criteria died of the disease [11].
Box 1. Weiss system.
High nuclear grade
Mitotic figures > 5/50 high-power fields
Atypical mitotic figures Eosinophilic or compact tumor cell cytoplasm (> 75% of tumor cells) Diffuse architecture (> 33% of tumor)
Necrosis (confluent necrosis) Venous invasion (smooth muscle in wall)
Sinusoidal invasion (no smooth muscle in wall)
Capsular invasion
The advantages of this scoring system are summarized as follows: i) the system is straightforward and relatively easy to use; ii) the scoring can be obtained only from histo- pathological findings based on examination of hematoxylin- eosin stained histological tissue slides, which made the wide application of this system possible; and iii) a good correlation has been demonstrated between results and clinical outcome of the patients in many institutions of many different countries.
There are a few case reports that suggest the limitations of this system, including that in which no pathologists were listed as co-authors [13]. In addition, as is true in any of the histological scoring systems, an evaluation done in poorly prepared pathology specimens, including those associated with delayed fixation, provides unreliable results in this criterion of Weiss and proper preparation of the specimens is a prerequisite for obtaining reasonable results. It is also true, however, that the tumors that did not behave in a malignant fashion in their postoperative course, including the cases of adrenocortical oncocytoma, were considered as adrenocortical carcinoma, although these adrenocortical oncocytomas may recur or metastasize in a long period of time [14,15]. However, this excellent scoring system is associated with the following limitations or disadvantages: i) subjectivity or lack of reproducibility of the findings, as may be true in any of the histopathological scoring systems [16,17]; ii) limitations of the scoring in the tumor groups, such as adrenocortical oncocytoma, as described above, and pediatric adrenocortical tumors, described in the following; and iii) relative insufficiency regarding the classification of carcinoma cases into high- and low-risk groups. It is true that Weiss reported that, among the nine histological factors in his criterion, mitotic activity, especially with atypical forms, and venous invasion were correlated best with metastasizing or recurring behavior of the resected adrenocortical tumors [11], but this does not necessarily represent the definitive subclassification of adrenocortical carcinoma groups into high- and low-risk groups. At this juncture, none of the histological or clinical evaluations
could reasonably contribute to predicting the subsequent postoperative course of the patients with adrenocortical carcinoma, and molecular and/or biological analysis, which may provide the relevant information, will be required in the management of these patients.
The subjectivity of this scoring may be most influenced by familiarity of the diagnostic pathologists involved [17], which could pose problems, considering that the number of pathologists in training who are interested in endocrine pathology appears to be universally decreasing. Assessment of nuclear atypia, mitotic activity, the presence or absence of coagulation necrosis and atypical mitosis, features regarding cytoplasm and venous invasion, can be relatively easily appreciated by well-qualified pathologists. However, as summarized in Figure 1A, B, great care should be taken not to misinterpret the carcinoma present adjacent to endothelium as vascular or sinusoidal invasion. Carcinoma cells that histopathologically protrude into vascular lumen covered by endothelium, as seen in Figure 1B, should not be interpreted ‘vascular invasion’. In addition, capsular invasion may pose diagnostic difficulty in diagnosis, and we interpret those completely penetrating the capsule (Figure 2A) or invading extensively in the capsule as seen in Figure 2B, or those associated with desmoplasia with inflammatory cells infiltration or activated fibroblasts, as positive for capsular invasion, although the latter is not frequent. Evaluation of architecture could certainly become subjective, and the application of silver stain, which outlines each group of tumor cells, may sometimes be of help in demonstrating the architectural abnormalities.
Practically, it is important to combine gross features including those described previously and the histological scoring systems above in order to reach the diagnosis of adrenocortical carcinoma. However, as outlined above in the limitations of applying the criteria of Weiss, even careful combination of available clinical, macroscopic and micro- scopic information made it difficult or at least complicated to differentiate adrenocortical carcinoma from adenoma in pediatric cases. In the authors’ experience, including the evaluation of consultation cases, adrenocortical tumors histologically diagnosed as carcinoma based on the criteria described above turned out to behave less aggressively compared with adult cases. This may be due to the fact that the tumor is more probably completely excised, or the intrinsic biological behavior of the tumor itself is less aggressive in children. However, the combination of gross features and histological criteria described above is still considered to be reasonably effective in making the diagnosis of malignancy in pediatric adrenocortical neoplasm. For example, Wieneke et al. proposed the histopathological criteria or scoring systems of pediatric adrenocortical carcinoma [18]. Features associated with an increased probability of a malignant clinical behavior included tumor weight (> 400 g), tumor size (> 10.5 cm), vena cava invasion, capsular and/or vascular invasion, extension into periadrenal
A.
B.
A.
B.
soft tissue, confluent necrosis, severe nuclear atypia, > 15 mitotic figures per 20 high-power fields, and the presence of atypical mitotic figures. Vena cava invasion, necrosis and increased mitotic activity (> 15 mitotic figures per 20 high- power fields) independently suggest malignant clinical behavior in multivariate analysis.
4. Molecular and cellular diagnosis of adrenocortical carcinoma in resected adrenal tumors
Recently, the application of cellular biology and molecular tools in cancer research has provided new information on human cancer biology. However, relatively rare frequency of adrenocortical carcinoma prevents investigators from drawing definitive conclusions about the biological significance of the results obtained from sporadic molecular and cellular studies reported in the literature. In addition, there are no established pre-malignant conditions in human adrenal cortex and the transition from adrenocortical adenoma to
carcinoma has not been well documented, in contrast to other human malignancies. Therefore, the possible significance or roles the molecular and cellular abnormalities detected in carcinoma patients in tumorigenesis or carcinoma development can be very difficult to evaluate and to apply to actual diagnosis and management of the patients. In addition, marked heterogeneity of both morphology and cell biological features, even among the same tumor specimens, made it nearly impossible to draw definitive conclusions regarding whether the findings obtained from one area of the carcinoma represent the features of the whole tumor, or, eventually, characteristics of the patients with adrenocortical carcinoma, compared with other malignancies. Therefore, those involved in the management of the patients with adrenocortical carcinoma must first realize when reading the articles of application of molecular and cellular studies to human adrenocortical carcinoma that their results are clinically of no value and/or significance even if meticulously and elegantly performed using sophisticated methods unless the findings are correlated with morphological features of the
carcinoma. Therefore, in this section, recent developments in molecular and cellular features of adrenocortical carcinoma will be summarized, with emphasis on the possibility of applying them to the evaluation of the differences between adrenocortical adenoma and carcinoma and/or of the biological or clinical behavior of the resected neoplasms as auxiliary diagnostic means.
4.1 DNA ploidy
As may be expected, adrenocortical neoplasms that recurred or metastasized were more often associated with DNA aneuploidy than those with no evidence of further disease during the postoperative follow-up period [19]. For example, seven of eight clinically confirmed adrenocortical carcinomas demonstrated DNA aneuploidy, whereas all adenomas were diploid by flow cytometry [20]. However, as in other malignancies, several studies also reported that 20 - 40% of adrenocortical adenomas have DNA aneuploidy and a small subset of carcinoma was diploid [20-23]. In addition, Camuto et al. demonstrated that there was no correlation among ploidy status and survival, and response to therapy [24]. Therefore, the value of DNA ploidy in determining biological behavior of resected adrenocortical behavior as a possible auxiliary means of evaluating adrenocortical neoplasms should require further investigation.
4.2 Parameters of cell proliferation
Cell kinetic information has become a valuable adjunct to histopathologically based tumor classification, to evaluation of response to therapy and of postoperative clinical course, and others [25]. Among the methods proposed to assess cell proliferation in resected tumor specimens submitted to diagnostic laboratories, immunohistochemical analysis of cell cycle-related antigens has advantages over other conventional methods, especially with respect to its cost performance, easy applicability and ability to obtain the proliferation rate of tumor cells. Among the cell cycle-related antigens applicable to immunohistochemistry, the monoclonal antibody Ki67, which recognizes a nuclear antigen present in all phases of the cell cycle, has been used most extensively in diagnostic pathology laboratories. The availability of MIB-1 with combination of antigen retrieval made it possible to perform Ki67 immunostaining in 10% formalin-fixed and paraffin-embedded materials (Figure 3). The Ki67 labeling index of adrenocortical carcinoma has been reported to be significantly higher than adrenocortical adenoma [26-28]. In the authors’ study of immunohistochemical evaluation of Ki67/MIB-1 in human adrenocortical neoplasms, 11 of 17 carcinomas demonstrated a labeling index of > 2.5, whereas none of the adenomas did [27]. Therefore, resected adrenocortical neoplasms of labeling index > 2.5 may represent adrenocortical carcinoma. Ki67 immunostaining is of value in differentiation between adrenocortical adenoma and carcinoma, and it would be reasonable to propose that it be incorporated in the histological evaluation of
adrenocortical neoplasms, especially histologically intermediate cases. However, as in any medical diagnostic methodology, this method is not perfect and has the following limitations that readers should recognize: the problems of inter- or intraobserver differences when applying Ki67 labeling index to surgical pathology differential diagnosis between adreno- cortical adenoma and carcinoma. This is a universal problem and has been experienced frequently at the time of evaluation of Ki67 immunostain of any resected neoplasms. This problem, which could become sometimes serious, is considered to be derived from the following two factors: i) uneven distribution of Ki67 immunoreactivity, that is, how many fields or cells it is necessary to count - this is largely due to poor fixation or preparation of the specimens and/or inert heterogeneity of the specimens; and ii) interpretation of weak nuclear immunoreactivity, that is, the criterion of which immunopositive cells should be counted as positive nuclear stain. In the authors’ evaluation of Ki67 immuno- reactivity in adrenocortical tumors, at least 10 fields were selected, and at least 500, preferably 1000, tumor cells were counted. Even when a computer image analyzer is used for evaluation of Ki67 immunohistochemistry, the selection of the fields for counting and the threshold of nuclear positivity can still pose problems. The well-established MIB-1 mono- clonal antibody in combination with reliable instrument such as automatic staining instruments provided relatively constant and reproducible results of immunohistochemistry. Therefore, at this juncture, to maximize this most cost- effective adjunctive method in differentiating between adrenocortical carcinoma and adenoma proper preparation of the resected specimens is necessary, that is, prompt and appropriate fixation.
4.3 Growth factors and their receptors
Overexpression and/or other abnormalities of various growth factors and their corresponding receptors have been demonstrated to be associated with aggressive biological behavior in many human malignancies. In human adrenal cortex and its various disorders, growth factors have been examined for their possible roles in modifying corticosteroids production and/or secretion through evaluation of adreno- cortical free cell preparation. However, abnormalities of growth factors have not necessarily been well studied in human adrenocortical carcinoma compared with other malignancies. Overexpression of transforming growth factor-o (TGF-a) and epidermal growth factor receptor (EGFR) has been demonstrated in adrenocortical carcinoma cases [29]. Subsequent studies also revealed that the status of EGFR immunoreacitivity was significantly more abundant in ACC than adrenocortical adenoma or non-pathological adrenal gland [29]. These findings of EGFR overexpression in human adrenocortcail carcinoma also suggest the potential target specific therapy in adrenocortical carcinoma patients.
Elevated expression of insulin-like growth factor (IGF)-II was reported in functioning adrenocortical carcinoma [30].
Very recently, Wilkin et al. demonstrated that induction of the phenotype of the fetal adrenal cortex by IGF-II overexpression and steroidogenesis as well as defective apoptosis may be the cause of pediatric adrenocortical tumors [31]. IGF-I overexpression was also reported in human adrenocortical carcinoma [32]. Inhibins and activins are dimeric proteins of the TGF-B super family. They were demonstrated to be present in human adrenal cortex and its disorders [33-35]. Munro et al. recently reported that loss of an inhibin-o subunit might be involved in the progression of adrenocortical carcinoma [33]. Fetsch et al. reported that immunocytochemistry of anti-o inhibin can reliably differentiate between adrenocortical and renal cell carcinoma in the specimens of fine needle aspiration [34,36]. However, Arola et al. reported no significant differences of inhibin-o expression between benign and malignant adrenocortical tumors and further investigations are required for clarifica- tion of possible roles of inhibins/activins in the develop- ment and progression of adrenocortical neoplasm [35]. Martinerie et al. reported that NOVH, which belongs to the CCNCCTGF/CYR61/NOV family of proteins, some of which have chemotactic, mitogenic, adhesive and angiogenic properties, could be involved in human adrenocortical tumor development [37]. Boccuzzi et al. also reported that the association between TGF-ß1 expression and active steroid secretion is lost in adrenocortical carcinoma [38]. Murray et al. demonstrated the decrement of a1 connexin 43 gap junctions, which suggests that an analysis of gap junction protein may be of use in the differential diagnosis between adrenocortical adenomas and carcinomas [39].
Vascular endothelial growth factor (VEGF) plays an important role in the regulation of tumor angiogenesis that is critical for tumor growth and metastasis [40]. A previous study reported that VEGF levels are significantly lower in adrenocortical adenoma than in adrenocortical carcinomas [41].
Adrenocorticotropic hormone (ACTH) and angiotensin-II are the most potent trophic fartors in normal adrenocortical cells [42-44]. Therefore, it is interesting to know the status of the receptors of these peptides in adrenocortical carcinoma compared with normal cortex and adrenocortical adenoma. Reincke et al. reported that the expression of ACTH receptor mRNA was lower in adrenocortical carcinoma than normal adrenal cortex and functioning adenoma and the correlation between ACTH receptor mRNA and P450scc mRNA detected in adrenocortical adenoma was not observed in the cases of adrenocortical adenoma [42]. Schubert et al. further demonstrated that angiotensin-II type 1 receptor expression was predominant in adrenocortical tumors and both angiotensin-II type 1 receptor and ACTH receptor were correlated with functional status of the tumors and lower than benign tumors [43]. In addition, altered expression patterns of angiotensin-II type 1 receptor were reported, by Pawlikowski et al. [44], in adrenocortical carcinoma using immunohistochemistry. These results all demonstrated that angiotensin-II and ACTH might play important roles in adrenocortical adenoma but not in carcinoma.
4.4 Cytogenetics and its potential applicability to differential diagnosis between adrenocortical carcinoma and adenoma
Etiology or the mechanism of tumorigenesis of human adrenocortical carcinoma is totally unknown, but susceptibility to adrenocortical carcinoma appears to be inherited in some individuals or families. For example, children with Beckwith- Wiedemann syndrome, a very rare hereditary growth disorder characterized by macroglossia, gigantism and omphalocele, tend to have an increased incidence of a number of tumors, including adrenal adenomas and adrenocortical carcinomas [45-48]. In this rare syndrome, genetic abnormalities affect the chromosome region (11p15), which contains several genes, including IGF-II , CDKN1C (cyclin-dependent kinase inhibitor p57kip2) and H19 (an untranslated mRNA) [49,50]. The p57kip2 gene is an embryonic cyclin-dependent kinase inhibitor that acts to regulate negatively cell proliferation and actively direct differentiation [50]. The H19 gene encodes a 2.3-kb non-coding mRNA, which is also strongly expressed during embryogenesis [50]. Genomic imprinting is a normal embryonic/fetal process that involves methylation of DNA regions leading to stable patterns of transcriptional gene activation or silencing [50]. Within this locus, IGF-II is normally expressed from the paternal allele owing to imprinting of the maternal copy, whereas CDKN1C and H19 are paternally imprinted and maternally expressed [50,51]. Individuals with Beckwith-Wiedemann syndrome often have uniparental paternal isodisomy (a form of loss of heterozygosity), that is, loss of the maternal locus with an accompanying gain of the paternal allele, then resulting in the remarkable overexpression of IGF-II with concomitant decrease in p57kip2 and H19 expression [50]. In addition, adrenocortical carcinoma is part of a constellation of tumors inherited in
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the sarcoma, breast, lung and adrenocortical carcinoma syndrome described by Li and Fraumeni [2] and Lynch et al. [53], called Li-Fraumeni syndrome, which is also very rare. The p53 gene, located on the 17p13.1 chromosomal segment, encodes the 393 amino acid tumor suppressor protein situated in the center of a network of signaling pathways that are essential for cell growth regulation and apoptosis induced by a diverse array of cellular stresses [50]. In the patient with Li-Fraumeni syndrome, tumors arise from germ-line loss of p53 include breast cancer, soft tissue sarcomas, brain tumors, osteosarcoma, leukemia and adrenocortical carcinoma [52,54]. These findings suggest, above all, the possibility of spontaneous transforma- tion of adrenocortical cells by spontaneous mutations of genomic DNA. However, it is also important for readers to recognize that only the minority of patients with this syndrome develop adrenocortical carcinoma and the clinical value of applying these genetic findings to differential diagnosis between adrenocortical carcinoma and adenoma still remains premature.
Various studies have suggested that loss of heterozygosity at loci on the short arm of chromosome 11 (11p) may be important in the pathogenesis of both benign and malignant adrenocortical neoplasm [55]. Yano et al. demonstrated that loss of alleles on chromosomes 11p, 13q and 17p was observed in both primary and metastatic adrenocortical carcinomas but not in adrenocortical adenomas [55]. A breakpoint of 11p13, as well as loss of heterozygosity of alleles on 11p15, has been recently reported in adrenocortical carcinoma cases [56]. Therefore, abnormalities of chromosome 11p are reasonably considered to be involved in tumorigenesis of adrenocortical carcinoma. Dohna et al. recently reported results of comparative genomic hybridization (CGH) analysis in human adrenocortical neoplasms [57]. Adenomas and carcinomas both demonstrated chromosomal imbalances, but several chromosomal gains, especially the high-level amplifications, were almost exclusively detected in adreno- cortical carcinoma. Zhao et al. also reported that the most frequent DNA copy number changes in adrenocortical carcinomas were losses of 1p21-31,2q,3p,39,6q,9p and 11q14-qter as well as gains of 17p,17q and 9q32-qter in their CGH study [58]. These authors postulated that oncogenes determining the early tumorigenesis of adreno- cortical tumors might exist on chromosome 17 [58]. Russell et al. reported that changes in chromosomes 3, 9 and X are early events in adrenocortical tumorigenesis, with increasing chromosomal instability with tumor progression [59]. These inconsistent results of CGH analysis re-emphasized heterogeneity of human adrenocortical neoplasm. These findings are also far from applying these factors in the differential diagnosis at this juncture.
Previously, germ-line mutations of the p53 tumor suppressor gene have been implicated in the etiology of this disorder [60]. The germ-line mutations detected in Li-Fraumeni syndrome appear to be clustered in exon 7 of
the p53 gene and have single-base substitutions resulting in amino acid changes, although a wide range of germ-line p53 mutations may be inherited [61]. Subsequent studies revealed that germ-line p53 mutations were also found in cancer- prone individuals who were not otherwise indicative of the Li-Fraumeni syndrome [60,62]. Therefore, it is interesting to know whether germ-line p53 mutations are present or not in sporadic adrenocortical carcinoma, which comprises the great majority of carcinoma cases. Wagner et al. reported that 3 of 6 children with adrenocortical carcinoma were found to carry germ-line p53 mutations in exons 5, 6 and 7, respectively [63]. Barzon et al. recently reported that mutations in the p53 gene are frequent in adrenocortical carcinomas [64]. However, it is also important to note that patients with adrenocortical carcinoma, possibly except for a specific pediatric case, are by no means prone to the development of other primary malignancies, and familial cases of adrenocortical carcinoma are rare. Therefore, the great majority of cases with sporadic adult adrenocortical carcinoma are considered not to harbor germ-line mutations of p53, but further investigations are needed for clarification. It is also important to note that p53 gene abnormality is one of the most common genetic alterations detected in human malignancies. Reincke et al. reported the relative low prevalence of p53 abnormalities, that is, 3 out of 11 cases demonstrated p53 abnormalities, although none of 5 adrenocortical cases demonstrated p53 abnormalities [65]. McNicol et al. reported that abnormal p53 expression did not appear to have any significant prognostic effects in carcinoma [66]. Therefore, in contrast to relatively close association of adrenocortical carcinoma with germ-line p53 mutations in some pediatric cases, p53 abnormalities do not appear to play important roles in the tumorigenesis or development of most adrenocortical carcinomas. Abnormalities of other oncogenes or tumor suppressor genes have not been studied in detail. Suzuki et al. reported altered intracellular localization of c-myc oncogene product in adrenocortical carcinoma, but further investigations are awaited to clarify the practical importance of the findings [20]. Gortz et al. reported that inactivating mutations of the MEN-1 tumor-suppressor gene appear not to play a role in the development of sporadic adrenocortical neoplasms [67]. Heppner et al. also reported that most of the seven adrenocortical carcinoma cases examined were associated with 11q13 loss of heterogeneity, in which the MEN-1 gene is located, but somatic MEN-1 mutation within the MEN-1-coding region was a rare event [68]. Nakazumi et al. reported the possible involvement of decreased expression of p27, a cell cycle inhibitor, in the biological behavior of adrenocortical neoplasms [26]. Pilon et al. demonstrated an important role of inactivation of the p16 tumor suppression gene in the pathogenesis of human adrenocortical tumor [69]. Hirano et al. reported the possible correlation between telomerase activity and biological behavior of adrenocortical neoplasms [70]. Mantovani et al. recently reported that the
four regulatory subunits (R1A, R1B, R2A and R2B) of protein kinase A were more highly expressed in the adrenal carcinoma than the normal adrenal cortex [71]. Babinska et al. demonstrated the statistically significant correlation between p53, p21, PCNA or Ki67 and the occurrence of metastases in adrenocarcinoma [72]. Barzon et al. reported that the expression levels of ERa and aromatase are significantly higher and the ERß level was lower in adrenal carcinoma compared with the normal adrenal cortex [73]. The results of these studies using molecular and cellular biological tools all pointed out the importance of the abnormal cell proliferation in the development and progression of adrenocortical carcinoma. However, it is also important to evaluate the properties of invasion and metastasis of adrenocortical neoplasms in assessing biological behavior of resected adrenocortical neoplasms, but little has been examined in this field. Further investigations in this field may contribute greatly to our understanding of adrenocortical neoplasms.
Activating mutations of the Wnt signaling pathway have been reported in numerous human malignancies. Recently, Tissier et al. reported more frequent abnormal cytoplasmic and/or nuclear accumulation of ß-catenin and genetic mutations of ß-catenin in adrenocortical carcinoma [74]. These results suggest the potential involvement of abnormalities of the Wnt signaling pathway in the biology of adrenocortical carcinoma and this pathway may become the potential target of specific therapy for adrenocortical carcinomas.
4.5 DNA microarray analysis of adrenocortical tumors
The recent advent of molecular biology techniques, especially DNA microarray techniques, have made it possible to identify the underlying genetic abnormalities through analyzing global profile gene expression associated with malignant tumors. When comparing the results of microarray analysis with clinicopathlogical parameters or phenotypes of the carcinoma cases, one could evaluate the significance of gene expression abnormalities among those detected in assay. Comprehensive expression profiling of tumors using DNA microarrays has been used for molecular classification, biomarker discovery and others involved in biological and clinical features of the tumors. Giordano et al. first applied DNA microarray technologies to human adrenocortical disorders [75]. They examined 11 adrenocortical carcinomas, 4 adrenocortical adenomas, 3 normal adrenal gland and 1 macronodular hyperplasia (MNH) using Affymetrix HG_ U95Av2 oligonucleotide arrays representing ~ 10,500 unique genes. The results demonstrated the presence of many genes whose expression was significantly higher in carcinoma than in adenoma, but increased expression of IGF2 was identified in 10 of 11 adrenocortical carcinoma cases (90.9%). Velázquez-Fernández et al. examined 7 carcinomas and 13 adenoma cases using a DNA microarray that consisted of 29,760 cDNA fragments spotted onto Ultra GAPS slides [76]. Among genes that were most significantly upregulated in carcinomas were two ubiquitin-related genes (USP4 and
UFD1L) and several insulin-like growth factor-related genes (IGF2, IGF2R, IGFBP3 and IGFBP6). Among genes that were most significantly downregulated in carcinomas were a cytokine gene (CXCL10), several genes related to cell metabolism (RARRES2, ALDH1A1, CYBRD1 and GSTA4) and the cadherin 2 gene (CDH2). Slater et al. reported DNA microarray analysis in 10 adrenocortical carcinomas and 10 adenomas using D-chips containing 11,540 DNA spots [77]. Many genes were upregulated in adrenocortical carcinoma, but the IGF2 gene was significantly upregulated in ACCs versus cortical adenomas after confirmation by RT-PCR analysis, which is usually required for the validation of the results by DNA microarray. Genes that were downregulated in adrenocortical tumors included chromogranin B and early growth response factor 1.
These results of the comparative DNA microarray results between adrenocortical carcinoma and adenoma highlight the potential significance of IGF-II overexpression in adrenocortical carcinoma both as a diagnostic marker and as a surrogate marker for target specific therapy towards IGF-II signal inhibition. Higher IGF-II expression in the carcinoma was reported to be associated with a more aggressive phenotype [78]. In addition, IGF-II overexpression was also reported to be associated with a higher risk for ACC recurrence [79]. Also, Schmitt et al. reported that the combination of IGF-II immunohistochemistry with MIB-1 index led to high sensitivity and specificity in detecting adrenocortical carcinomas in the study of 17 carcinomas and 22 adenomas [80].
5. Conclusion
An evaluation of macroscopic features including the determina- tion of the areas for submitting to further evaluation and application of histological scoring systems, especially the criteria of Weiss, are still the most effective and cost-effective methodologies in discerning malignancy in resected adreno- cortical neoplasm. However, it is necessary for those involved in the diagnosis to become familiar with the system, aware of its limitations, such as in its application to pediatric neoplasms or to adrenocortical oncocytoma, and the require- ment of appropriate preparation of the specimens for diagnosis. Among auxiliary methods, the Ki67/MIB-1 labeling index and evaluation of IGF-II overexpression in the tumor tissues may be considered to be worth incorporating into practice.
6. Expert opinion
When evaluating the resected adrenocortical neoplasms, first it is necessary to determine whether the tumors are malignant or not and, if malignant, how aggressive they will be, including the possible response to various antineoplastic agents, including op’-DDD in the cases of adrenocortical carcinoma. Translational research in general should be
directed towards the areas in which conventional diagnostic methods are insufficient to provide satisfactory information as to the management of the patients. Despite some drawbacks or limitations as outlined in this review, an application of the criteria of Weiss on histological sections is a quite cost-effective diagnostic method and could provide reasonably satisfactory information as to discerning malignancy in resected specimens. With combination of the Ki67/MIB-1 labeling index, which is also a cost-effective laboratory diagnostic method, there will be little room left for incorporating molecular or other sophisticated methodologies into discerning malignancy itself in the tumor. However, it is also true that among the patients of adrenocortical carcinoma, some undergo a markedly aggressive postoperative clinical course, which may require some target-specific therapies other than conventional chemotherapy. In addition, op’-DDD has been introduced as the specific adrenocorticolytic therapeutic agent, but only 2 - 30% of the patients responded to the treatment and the treatment itself was associated with marked side effects. At this juncture, there are no established clinical or histological
methods to predict postoperative course or biological behavior and the response to op’-DDD or other therapy in the patients with adrenocortical carcinoma. Therefore, molecular analysis of adrenocortical carcinoma in the near future should be targeted towards this area. The results of DNA microarray from some laboratories all pointed out the overexpression of IGF-II in adrenocortical carcinoma, which suggests the importance of IGF-II-related intracellular signal pathways in adrenocortical carcinoma. Therefore, the development of target-specific therapy towards inhibition of IGF-II signals could provide clinical benefits to the patients in the near future. If this is the case, an evaluation of IGF-II overexpression in the resected specimens may become a prerequisite for the management of the patients with adrenocortical carcinoma as in HER2/neu evaluation in breast carcinoma patients.
Declaration of interest
The authors state no conflict of interest and have received no payment in preparation of this manuscript.
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