Clinical Research
Discerning Malignancy in Resected Adrenocortical Neoplasms
Hironobu Sasano, MD, Takashi Suzuki, MD, and Takuya Moriya, MD
Abstract
Differential diagnosis between adenoma and carcinoma in resected human adrenocorti- cal neoplasms may be one of the most problematic and difficult areas of surgical pathol- ogy practice. This is especially true in cases of relatively small adrenocortical tumors not associated with obvious signs of malignancy such as necrosis and/or hemorrhage. In addi- tion, the numbers of these small adrenocortical neoplasms are increasing owing to the widespread application of sophisticated computed tomography and/or magnetic reso- nance imaging scans. No single parameter can be effective in this differential diagnosis of resected adrenocortical tumor. Histopathologic evaluation using a multivariate scor- ing system is considered most effective in discerning malignancy and biologic behavior of resected adrenocortical neoplasms. Molecular and cellular findings of adrenocortical car- cinoma have been inconsistent except for the increased cell proliferation associated with adrenocortical malignancy. Therefore, an assessment of neoplastic cell proliferation using immunostains of cell cycle-associated nuclear antigen such as Ki-67 is the only useful auxilliary method of evaluating malignancy in resected adrenocortical neoplasms at present.
Key Words: Adrenal; cortex; carcinoma; adenoma; differential diagnosis; pathology.
Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.
Address correspondence to Dr. Hironobu Sasano, Department of Pathology, Tohoku University School of Medicine, 2-1 Seiryou-machi, Aoba-ku, Sendai, Japan 980-0872. E-mail: hsasano@patholo2.med.tohoku.ac.jp
Endocrine Pathology, vol. 12, no. 4, 397-406, Winter 2001 @ Copyright 2001 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/01/12:397-406 $12.50
Introduction
The most important and critical point in adrenocortical pathology is the differ- ential diagnosis between adrenocortical adenoma and carcinoma. In this review, we briefly summarize gross and pathologic findings pertinent to differential diagno- sis, especially histopathologic differential diagnostic criteria, and summarize recent cellular and molecular findings that may contribute to differential diagnosis of adrenocortical malignancy.
Macroscopic
When evaluating malignancy of resected adrenocortical neoplasms, macroscopic observation of the specimen submitted to
diagnostic pathology laboratories is extremely important. The first important factor is the weight of the tumor. There- fore, the weight of the neoplasm should be determined as carefully as possible when evaluating adrenocortical neoplasm. In our experience, of 66 cases of tumor weighing >100 g, the tumor comprised 93% of the carcinoma but only 6% of the adenoma. Tang and Gray, [1] reported that all corti- cal tumors weighing >95 g were malignant, whereas tumors <50 g were benign (the average weight of the tumor is 705 g rang- ing from 96 to 2460 g). Slooten et al. [2] reported that in their series, only tumors weighting >150 g metastasized. However, note that small adrenocortical tumor can metastasize and some large tumors do not. A tumor reported by Gandour and Grizzle
[3] weighed only 40 g and measured only 4 cm in greatest dimension but metastasized 3 yr following bilateral adrenalectomy. We have also seen a right adrenocortical tumor weighing only 19 g and measuring 4 x 4 x 3 cm with Cushing syndrome that recurred in 3 yr and 2 mo following surgery [4]. On the other hand, Hough et al. [5] reported that a tumor weighing 1800 g did not metastasize. Therefore, the weight of a tumor is important in evaluating malig- nancy of adrenocortical neoplasms, but the weight itself is not a reliable prognostic indicator of the resected adrenocortical tumor.
The next important factor is the mac- roscopic features of the cut surface of the tumor. In our experience, hemorrhage and necrosis are rarely observed in adrenocor- tical adenoma. Necrosis is sometimes associated with cystic degeneration. The presence of necrosis and hemorrhage, therefore, strongly indicates a diagnosis of adrenocortical carcinoma. However, it is also true that many adrenocortical carci- nomas 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. Note also that foci of intratumoral fibrosis and myxomatous degeneration [6] can be seen in both adenoma and carcinoma, and that adreno- cortical carcinoma may be well circum- scribed and encapsulated. The color of the cut surface of viable parts of adrenocor- tical neoplasms is not a reliable indicator of adrenocortical carcinoma. Carcinoma may be tan, yellow, or yellow-orange, but a homogeneous black cut surface, as observed in black pigmented adenoma, is rarely observed in adrenocortical carcinoma.
Histologic Differentiation Between Adrenocortical Adenoma and Carcinoma
It is true that a large number of adreno- cortical carcinomas are associated with the characteristic gross features we have described, including large size, necrosis, and hemorrhage, and do not usually pose diagnostic problems. However, an increas- ing number of small adrenocortical neo- plasms have been discovered with the development of computed tomography and magnetic resonance imaging scans. Therefore, adrenocortical carcinomas not associated with these ominous macroscopic features have recently increased in num- ber. The distinction of these “well-differ- entiated” adrenocortical carcinomas from adenoma may be one of the greatest diag- nostic difficulties in surgical pathology practice. There is no single histologic cri- terion that can reliably differentiate adrenocortical carcinoma from adenoma like capsular or vascular invasion of thy- roid follicular carcinoma. Only the systems that evaluated multiple histologic and/or nonhistologic criteria of the resected cases can provide reliable histologic diagnosis.
Three different histologic scoring systems have been proposed by various investigators [2,5,7,8], and all are equally useful for predicting clinical outcome of the patients with resected adrenocortical neoplasms. These systems are summarized in Tables 1-3. Hough et al. [5] proposed 12 criteria, 7 histologic, and 5 nonhistologic, of predicting clinical outcome of patients by studying 41 cases of adrenocortical tumors. A numeric value for these 12 criteria was determined by employing a modified Bayes’ theorem for predicting the possibility of metastasis. When assessing an individual case, these numeric values are combined and the histologic and
Table 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
| Histologic criteria | Numeric value |
|---|---|
| Extensive regressive changes | 5.7 |
| Loss of normal structure | 1.6 |
| Nuclear atypia | 2.1 |
| Nuclear hyperchromasia | 2.6 |
| Abnormal nucleoli | 4.1 |
| Mitotic activity (>2/10 high-power fields) | 9.0 |
| Vascular or capsular invasion | 3.3 |
| Table 3. Hough System | |
|---|---|
| Criteria | Numeric value |
| Diffuse growth pattern | 0.92 |
| Vascular invasion | 0.92 |
| Tumor cell necrosis | 0.69 |
| Broad fibrous bands | 1.00 |
| Capsular invasion | 0.37 |
| Mitotic index (>1/10 high-power fields) | 0.60 |
| Pleomorphism | 0.39 |
| Nonhistologic | |
| Tumor mass (>100 g) | 0.60 |
| Urinary 17-ketosteroids (10 mg/[g creatinine-24 hr]) | 0.50 |
| Response to ACTH (17-hydroxysteroids increased two times after 50 µg of IV ACTH)ª | 0.42 |
| Cushing syndrome with virilism, virilism alone, or no clinical manifestations | 0.42 |
| Weight loss (>10 lb/3 mo) | 2.00 |
| aACTH, adrenocorticotropic hormone. | |
nonhistologic indices are subsequently determined. The combination of histologic and nonhistologic indices was reported to effectively differentiate adrenocortical
carcinoma from adenoma [5]. This crite- rion was very useful in predicting subse- quent biologic behavior of resected adrenocortical tumor once familiar with the system. In addition, the inclusion of clinical findings is considered to enhance the reliability of the diagnosis. Neverthe- less, this system may be limited by the requirement of clinical findings, although results of these findings must be available for pathologists, and possibly the relatively complicated numeric processes.
Slooten et al. [2] developed a similar scoring system using seven histologic cri- teria of the resected neoplasms (Table 2) with assigned numeric value for differen- tiating adrenocortical carcinoma from adenoma. The numeric values for these severe histologic parameters described in Table 2 were subsequently combined and a histologic index for their case was obtained. A histologic index of more than eight is considered to correlate with aggres- sive biologic behaviors. When we retrospec- tively applied Slooten et al.’s [2] criteria to resected adrenocortical tumor, some adrenocortical adenomas were erroneously diagnosed as carcinoma. However, the evaluation of these histologic features is straightforward, and it is relatively easy to apply this system in histologic diagnosis.
In 1984, Weiss [7] proposed nine histo- logic criteria (Table 1), important in evalu- ating adrenocortical malignancy. Weiss subsequently lowered the threshold for adrenocortical malignancy from four to three histologic criteria because 20 of 23 patients who fulfilled three histologic criteria died of disease [8]. The system is straightforward and relatively easy to use, and a good correlation can be detected between results and clinical outcome of the patients. However, it is also true that the tumors that did not behave in a malignant
fashion in their postoperative course, including the cases of adrenocortical oncocytoma [9], were considered as adrenocortical carcinoma, although these adrenocortical oncocytomas may recur or metastasize over a long period of time. In addition, among these nine criteria, we experienced that nuclear grade, architec- ture, and cytoplasm were likely to be sub- jective; that is, the interobserver differences were relatively marked unless observers were well informed prior to histologic examination of adrenocortical tumor.
In general, it is important to combine gross features including those described previously and the histologic scoring sys- tem described earlier in order to reach a diagnosis of adrenocortical carcinoma. The value of histologic criteria and gross fea- tures of the resected specimens for differ- entiating adrenocortical carcinoma from adenoma in pediatric cases is more com- plicated than in adult cases. In our experi- ence, in pediatric cases adrenocortical tumors histologically diagnosed as carci- noma based on the criteria described turned out to behave less aggressively com- pared with adult cases. This is possibly because the tumor is more likely com- pletely excised or the intrinsic biologic behavior of the tumors itself is less aggres- sive in children. However, the combina- tion of gross features and histologic criteria we have described is still considered to be reasonably effective in making a diagnosis of malignancy in pediatric adrenocortical neoplasms.
Molecular and Cellular Features of Adrenocortical Carcinoma
Recently, the application of molecular and cellular biology and molecular tools in cancer research have yielded a new dimension in our understanding of human
cancer. However, molecular and cellular features of adrenocortical carcinoma are not necessarily well studied compared to other human malignancies. The relatively rare frequency of adrenocortical carcinoma prevents investigators from drawing defini- tive conclusions about the biologic signi- ficance of the results obtained from molecular and cellular studies. In addition, there are no established premalignant con- ditions in human adrenal cortex, and the transition from adrenocortical adenoma to carcinoma has not been well documented. Therefore, the possible significance or roles of the molecular and cellular abnormali- ties detected in patients with carcinoma in tumorigenesis or the development of carcinoma can be quite difficult to evalu- ate. Furthermore, human adrenocortical carcinomas are markedly heterogeneous in morphology and biologic function even within the same tumor. Thus, it is impor- tant to note that molecular and cellular features of human adrenocortical carci- noma are clinically of no value or signifi- cance even if studies are meticulously and elegantly conducted unless the findings are correlated with morphologic features. In the next sections, we therefore summarize recent developments in molecular and cellular features of adrenocortical carci- noma with emphasis on the possibility of applying them to the evaluation of the differences between adrenocortical adenoma and carcinoma and/or of the biologic behavior of the resected neoplasms as auxiliary diagnostic means.
DNA Content
Adrenocortical neoplasms that recurred or metastasized were reported to be more likely to demonstrate DNA aneuploidy than those showing no evidence of further disease during the postoperative follow-up period [10]. We also reported that seven
of eight adrenocortical carcinomas dem- onstrated DNA aneuploidy while all adenomas were diploid by flow cytometry [11]. However, a number of studies also reported that 20-40% of adrenocortical adenomas have DNA aneuploidy and that a small subset of carcinomas was diploid [12-14]. In addition, Camuto et al. [15] demonstrated that there was no correlation among ploidy status and survival, response to therapy, or steroid hormone production in adrenocortical neoplasms. Therefore, the value of DNA ploidy in determining the biologic behavior of resected adrenocorti- cal carcinomas is still in dispute, and fur- ther studies are required to establish it as a possible auxiliary means of evaluating adrenocortical neoplasms.
Cell Proliferation
Information about cell kinetics is becoming a valuable adjunct to histopatho- logically based tumor classification [16]. Among the various methods used to assess cell proliferation or cell kinetics in surgical pathology specimens submitted to diagnostic pathology laboratories, immu- nohistochemical analysis of cell cycle- related antigens has advantages over other conventional methods.
The monoclonal antibody Ki67 is con- sidered to recognize a nuclear antigen present in all phases of the cell cycle while the proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase 8 and is associated with the late G1- and S-phase of the cell cycle. The avail- ability of MIB-1 with a combination of antigen retrieval made it possible to per- form Ki67 immunostaining in 10% formalin-fixed and paraffin-embedded materials. The Ki67 labeling index (LI) of adrenocortical carcinoma was reported to be significantly higher than adrenocorti- cal adenoma by our group [13] and
Goldblum et al. [17]. On the other hand, the PCNA LI was not significantly differ- ent between adrenocortical adenoma and carcinoma [11,17], as was reported in other human malignancies. In our study of immunohistochemical evaluation of Ki67 in human adrenocortical neoplasms, 11 of 17 carcinomas had an LI of >2.5, whereas none of the adenomas did [18]. Therefore, resected adrenocortical neoplasms with an LI of >2.5 may represent adrenocortical carcinoma. Consequently, Ki67 immuno- staining is of value in differentiation between adrenocortical adenoma and carcinoma and may be incorporated in the histologic evaluation of adrenocortical neoplasms, especially histologically inter- mediate cases.
However, as is well known, inter- or intraobserver differences can become prob- lems when applying the LI of Ki67 to sur- gical pathology differential diagnosis between adrenocortical adenoma and car- cinoma. These differences can frequently be experienced through evaluation of Ki67 immunostain of resected neoplasms in vari- ous laboratories because of uneven distri- bution of Ki67 immunoreactivity, (i.e., how many fields are necessary to count) and interpretation of weak nuclear immu- noreactivity (i.e., the threshhold of posi- tivity when the LI of Ki67 is applied to resected adrenocortical carcinoma). In our laboratory, we select at least 10 fields and count at least 500, preferably 1000, tumor cells. Even when we use a computer image analyzer to evaluate the immunohis- tochemistry of Ki67, selection of the fields for counting and the threshhold of nuclear positivity can still be a problem.
Growth Factors
Overexpression and/or other abnormali- ties of various growth factors have been demonstrated to be associated with aggres-
sive biologic behavior in many human malignancies. In human adrenal and adrenocortical disorders, growth factors have been examined for their possible roles in modifying corticosteroid production and/or secretion through evaluation of the preparation of adrenocortical free cells. However, abnormalities of growth factors have not necessarily been well studied in human adrenocortical carcinoma, com- pared to other malignancies. Recently, overexpression of transforming growth factor-a (TGF-a) and epidermal growth factor receptor was demonstrated in adrenocortical carcinoma cases [19]. Elevated expression of insulin-like growth
factor-2 (IGF-2)was reported in function- ing adrenocortical carcinoma [20]. Overexpression of IGF-1 was also reported in human adrenocortical carcinoma [21].
Inhibins and activins are dimeric pro- teins of the TGF-ß superfamily. They were demonstrated to be present in human adrenal cortex and its disorders [22-24]. Munro et al. [22] recently reported that loss of inhibin a-subunit may be involved in the progression of adrenocortical carci- noma. However, Arola et al. [24] more recently reported no significant differences of inhibin a expression between benign and malignant adrenocortical tumors, and further investigations are required to clarify the possible roles of inhibins/activins in the development and progression of adreno- cortical neoplasms.
Cytogenetics
The etiology or the mechanism of tum- origenesis of human adrenocortical carci- noma is unknown, but it appears that susceptibility to adrenocortical carcinoma appears to be inherited in some individu- als or families. Children with Beckwith- Wiedemann syndrome, a very rare growth
disorder characterized by macroglossia, gigantism, and omphalocele [25,26], have an increased incidence of several tumors, including adrenal adenomas and adreno- cortical carcinomas [27,28]. Genetic abnormalities affect the chromosome region 11p15 in these patients [29]. Abnormalities of the TP53 gene have also been reported in these patients. Adreno- cortical carcinoma is part of a constella- tion of tumors inherited in the sarcoma, breast, lung, and adrenocortical carcinoma syndrome described by Li and Fraumeni [30] and Lynch et al. [31], called Li-Fraumeni syndrome, which is also very rare. These observations suggest that some carcinomas, although few, are considered to occur as a result of spontaneous transformation of adrenocortical cells by spontaneous muta- tions of genomic DNA.
Various studies have suggested that loss of heterozygosity at loci on the short arm of chromosome 11 (11p) may be impor- tant in the pathogenesis of both benign and malignant adrenocortical neoplasms [32]. Yano et al. [32] demonstrated that loss of alleles on 11p, 13q, and 17p was observed in both primary and metastatic adrenocor- tical carcinomas but not in adrenocortical adenomas. A breakpoint of 11p13, as well as loss of heterozygosity of alleles on 11p15, has recently been reported in cases of adrenocortical carcinoma [33]. Therefore, abnormalities of chromosome 11p are reasonably considered to be involved in tumorigenesis of adrenocortical carcinoma.
Dohna et al. [34] recently reported results of comparative genomic hybridiza- tion analysis in human adrenocortical neo- plasms. Adenomas and carcinomas both demonstrated chromosomal imbalances, but several chromosomal gains, especially the high-level amplifications, were almost exclusively detected in adrenocortical car-
cinoma. Zhao et al. [35] also reported that the most frequent DNA copy number changes in adrenocortical carcinomas were losses of 1p21-31, 2q, 3p, 3q, 6q, 9p and 11q14-qter as well as gains of 17p, 17q, and 9q32-qter in their comparative genomic hybridization (CGH) study. They postu- lated that oncogenes determining the early tumorigenesis of adrenocortical tumors may exist on chromosome 17 [35]. Russell et al. [36] reported that changes in chro- mosomes 3, 9, and X are early events in adrenocortical tumorigenesis, with increasing chromosomal instability with tumor progression. These inconsistent results of CAH analysis reemphasized the heterogeneity of human adrenocortical neoplasms.
Recently germ-line mutations of the p53 tumor suppressor gene have been implicated in the etiology of Li-Fraumeni syndrome [37]. 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 result- ing in amino acid changes, although a wide range of germ-line p53 mutations may be inherited [38]. Subsequent studies revealed that germ-line p53 mutations that were not otherwise indicative of the Li-Fraumeni syndrome were also found in cancer-prone individuals [37,39]. Therefore, it is important 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. [40] recently reported that three of six children with adrenocortical carcinoma were found to carry germ-line p53 mutations in exons 5, 6, and 7, respectively. However, note that patients with adrenocortical carcinoma, with the possible exception of pediatric cases, are by
no means prone to the development of other primary malignancies, and familial cases of adrenocortical carcinoma are rare. Thus, the most cases with sporadic adult adrenocortical carcinoma are considered not to harbor germ-line mutations of p53, but this needs to be clarified by further investigations.
Abnormality of the p53 gene is also one of the most common genetic alterations detected in human malignancies, which makes it important to know whether or not p53 abnormalities are detected in adreno- cortical carcinoma tissues. Reincke et al. [41] reported the relative low prevalence of p53 abnormalities. In their study, 3 of 11 cases demonstrated p53 abnormalities. although 0 of 5 adrenocortical cases demonstrated p53 abnormalities. McNicol et al. [42] reported that abnormal p53 expression did not appear to have any sig- nificant prognostic effects in carcinoma. We also could not detect any p53 abnor- malities including overexpression of p53 nuclear protein and p53 DNA mutations in 10 sporadic adult adrenocortical carci- noma cases. Therefore, in contrast to rela- tively close association of adrenocortical carcinoma with germ-line p53 mutations in some pediatric cases, p53 abnormalities do not appear to play important role in the tumorigenesis or development of the majority of adrenocortical carcinomas.
Abnormalities of other oncogenes or tumor suppressor genes have not been stud- ied in detail. Suzuki et al. [11] reported altered intracellular localization of c-myc oncogene product in adrenocortical carci- noma, but further investigations are needed to clarify the practical importance of the findings. Gortz et al. [43] reported that inactivating mutations of the multiple endocrine neoplasia 1 (MEN 1) tumor suppressor gene appear not to play role in
the development of sporadic adrenocorti- cal neoplasms. Heppner et al. [44] also reported that the majority of seven adreno- cortical carcinoma cases examined was associated with 11q13 loss of heterogene- ity, in which MEN 1 gene is located, but somatic MEN 1 mutations within the MEN 1-coding region were rare events. Nakazumi et al. [45] reported the possible involvement of decreased expression of p27, a cell-cycle inhibitor, in the biologic behavior of adrenocortical neoplasms. Hirano et al. [46] reported the possible correlation between telomerase activity and biologic behavior of adrenocortical neo- plasms.
Conclusion
The results of these studies employing molecular and cellular biologic tools all pointed to the importance of abnormal cell proliferation in the development and pro- gression of adrenocortical carcinoma. However, it is also important to evaluate the properties of invasion and metastasis of adrenocortical neoplasms, in assessing biologic behavior of resected adrenocorti- cal neoplasms, but little has been examined in this field. Further investigations likely will contribute greatly to our understand- ing of adrenocortical neoplasms.
Acknowledgment
This article was presented in part at the Endocrine Pathology Society session at the USCAP Meeting, Atlanta, A, March 3, 2001.
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