EPS Proceedings
Recent Advances in Histopathology and Immunohistochemistry of Adrenocortical Carcinoma
Hironobu Sasano, MD, PHD, Takashi Suzuki, MD, PHD, and Takuya Moriya, MD, PHD
Abstract
Discerning malignancy in resected adrenocortical neoplasms can pose diagnostic diffi- culty. Macroscopic examination is the first important step toward diagnosis and should include accurate measurement of weight and dimension of the specimens and descrip- tion of the cut surface of the tumors. It is also important to sample the specimens for histological diagnosis near foci of hemorrhage and/or necrosis. Histological scoring sys- tems evaluating multiple parameters, especially the criteria of Weiss, have been shown to be reliable in differential diagnosis between adrenocortical adenoma and carcinoma. A tumor is defined as adrenocortical carcinoma when three or more of the following criteria are met; (1) high nuclear grade, (2) mitotic rate six or more per 50 high power fields, (3) atypical mitosis, (4) clear cells less than 25%, (5) a diffuse architecture pattern in more than one-third of the tumor, (6) confluent necrosis, (7) venous invasion, (8) sinu- soidal invasion, and (9) capsular invasion. The criteria are relatively straightforward and considered the most effective standard for diagnosis of adrenocortical malignancy. How- ever, great care should be taken in applying the criteria to histological evaluation of two relatively rare and peculiar adrenocortical tumors, adrenocortical oncocytoma and pedi- atric adrenocortical neoplasms. At this juncture, ancillary biological or molecular markers are of little practical value in terms of differential diagnosis between adrenocortical adenoma and carcinoma but tumors with MIB1 or Ki-67 labeling index more than 2.5 may be considered malignant. Prognostic markers of adrenocortical carcinoma have not been established other than complete respectability of the tumor. There are also no surrogate markers for predicting response to therapy with Mitotane, an adrenolytic agent. It sometimes is important for surgical pathologists to differentiate adrenocortical carci- noma from metastatic malignancies of other sites. An immunohistochemical evaluation of adrenal 4 binding protein (Ad4BP) or SF-1, a transcription factor of all steroidogenesis, can aid in this differential diagnosis because nuclear immunoreactivity for this transcrip- tion factor is relatively specific to steroid producing cells.
Key Words: Adrenal; cortex; carcinoma; pathology; histology; immunohistochemistry; diagnosis.
Department of Pathology, Tohoku University School of Medicine and Tohoku University Hospital, 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. 17, no. 4, 345-354, Winter 2006 @ Copyright 2006 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046-3976/1559-0097 (Online)/ 06/17:345-354/$30.00
Introduction
The most pivotal and critical point in adrenocortical pathology is the histopatho- logical differential diagnosis between adrenocortical adenoma and carcinoma.
In addition, when the adrenal lesions are considered malignant, it is important to differentiate adrenocortical carcinoma from metastatic or invasive malignancies from other sites in order to determine appropriate management of the patients.
This brief review summarizes recent advances in histopathological and immuno- histochemical approaches useful in differ- ential diagnosis between adrenocortical adenoma and carcinoma and in dif- ferentiating adrenocortical primary from metastasis.
Differential Diagnosis Between Adrenocortical Adenoma and Carcinoma
The following summarizes what surgi- cal pathologists should be aware of when evaluating resected specimens for differen- tial diagnosis between adrenocortical adenoma and carcinoma.
Gross Examination
Gross examination is important in the evaluation of adrenocortical malignancy, as in other sites. The important steps in macroscopic examination follow.
The Exact Measurement of Tumor Weight
When receiving the specimens of resected adrenal mass, the first important step is the precise measurement of tumor weight after removing the adjacent adipose or other soft tissues. Weight of the tumor is a very important factor in differential diagnosis of an adrenal mass. For instance, among 66 cases of adrenocortical neo- plasms with full blown Cushing’s syndrome operated between 1954 and 1985 at Tohoku University Hospital, Sendai, Japan, tumors weighing more than 100 g comprised 93% of carcinoma but only 6% of the adenoma [1,2]. However, it is also important to note that small adrenocorti- cal tumors can metastasize and some large tumors do not. For instance, the tumor reported by Gondour and Grizzle weighed only 40 g, and measured only 4 cm in great-
est dimension but metastasized 3 yr fol- lowing bilateral adrenalectomy [3]. We also experienced a right adrenocortical tumor weighing only 19 g and measuring 4 x 4 x 3 cm with Cushing’s syndrome which recurred in 3 yr and 2 mo following cura- tive surgery [2]. On the other hand, Hough et al. reported that a tumor weighing 1800 g did not metastasize [4]. Therefore, while tumor weight is very important, weight alone is not a reliable indication of malig- nancy. In addition to tumor weight, gross description should include measurement of precise dimensions of the neoplasm and should indicate whether or not the tumor is circumscribed or encapsulated.
Description of the Cut Surface
The next important item is precise description of the cut surface of the tumor after carefully halving the resected speci- men to document the presence or absence of foci of hemorrhage and necrosis, which are rarely detected in adrenocortical adenoma. Necrosis is sometimes associated with cystic degenerative changes. The pres- ence of both necrosis and hemorrhage, is strongly suggestive of adrenocortical car- cinoma. However, many adrenocortical carcinomas do not exhibit these ominous findings. It is also critical to sample the specimens from areas adjacent to foci of necrosis and hemorrhage when grossing the specimens. We experienced a case in which histological diagnosis of adrenocortical malignancy was made only in samples obtained from these areas and other areas did not show any features of carcinoma. Foci of intratumoral fibrosis or hyaliniza- tion can be seen in both adenoma and car- cinoma. Adrenocortical carcinoma may be well-circumscribed and encapsulated. The color of the cut surface of viable parts of adrenocortical neoplasms is not a reliable indicator of adrenocortical carcinoma.
Carcinoma can be tan, yellow, or yellow orange. However, a homogeneous black cut surface, as observed in black pigmented adenoma, or homogeneous golden yellow appearance as observed in aldosteronoma is rarely detected in adrenocortical carcinoma.
Histological Diagnosis
In the past, the great majority of adreno- cortical carcinoma exhibited characteristic gross features described above including large size, necrosis, and hemorrhage and did not usually pose diagnostic problems even at the time of gross examination. However with the recent advent and widespread application of sensitive and sophisticated imaging analysis including computed tomography (CT) and nuclear magnetic resonance (NMR), adrenal masses as small as 0.3 cm can now be clini- cally detected without hormonal abnor- malities. These masses are termed, adrenal incidentalomas and the management of affected patients, including the decision whether the adrenal should be removed, can be an important challenge for clini- cians. Adrenocortical carcinomas not associated with ominous macroscopic fea- tures have recently increased in number. The differential diagnosis of these “well- differentiated” adrenocortical carcinomas from adenomas poses one of the greatest diagnostic difficulties in surgical pathology practice at present. There are no established single histological criteria like capsular or vascular invasion of thyroid follicular car- cinoma that can reasonably differentiate well differentiated adrenocortical carci- noma from adenoma. Only systems that evaluate multiple histological and/or non- histological parameters can lead to reliable histological diagnosis. Among these sys- tems, the histological criteria proposed by Weiss in 1984 are currently the most widely employed. The Weiss criteria are composed
of the followings: (1) High nuclear grade; (2) mitotic rate six or more per 50 high power field; (3) atypical mitosis; (4) clear cells less than 25% of the tumor; (5) a dif- fuse architecture pattern in more than one- third of the tumor; (6) confluent and coagulative necrosis; (7) venous invasion; (8) sinusoidal invasion; and (9) capsular invasion [5]. Originally a tumor was con- sidered adrenocortical carcinoma when four or more of the criteria were met [5]. However, Weiss subsequently lowered the threshold for adrenocortical malignancy from four to three histologic criteria because 20 of 23 patients with tumors that fulfilled three histologic criteria died of disease [6]. When we retrospectively applied the criteria of Weiss to resected adrenocortical tumors, the system was found to be straightforward and relatively easy to use, and a good correlation was detected between results and clinical out- come of the patients including the tumor of 19 g described previously [2]. The fact that the criteria are solely histological find- ings has made it possible to apply them to many institutions all over the world.
However, among these nine criteria out- lined in Fig. 1, we experienced that “nuclear grade,” which is based on the nuclear grade of renal cell carcinoma, “architecture,” whose definition is somewhat vague (i.e., diffuse architecture can be difficult to interpret), and “cytoplasm,” in which definition of clear and compact cells could vary among pathologists, were likely to be subjective, i.e., interobserver differences were relatively marked unless observers were well-informed prior to histological examination of adrenocortical tumors or highly experienced in adrenocortical pathology. In addition, it has recently been demonstrated that great care should be taken in application of the Weiss criteria to differential diagnosis in two distinctive types of adrenocortical lesions, i.e., adreno-
cortical oncocytoma and pediatric adreno- cortical carcinoma.
Adrenocortical Oncocytoma
We first reported adrenocortical onco- cytoma in 1991 [7]. Histological features of adrenocortical oncocytoma are essen- tially similar to oncocytoma arising in other parts, i.e., abundant eosinophilic cytoplasm filled with numerous mitochondria in their cytoplasm (Fig. 1). This tumor is in gen- eral large, weighing up to more than 1000 g, and is usually associated with marked nuclear atypia such as large or bizarre nuclei and prominent nucleoli in histological examination (Fig. 1). The absence of lipid in cytoplasm of tumor cells and relatively large size of the tumor usually resulted in clinical or radiological diagnosis of adreno- cortical malignancy. In histological exami- nation of the resected specimens, nearly all cases of adrenocortical oncocytoma met at least two of the criteria of Weiss as described above. It is possible that adreno- cortical oncocytomas may recur or metas- tasize after a long period of time but at this juncture no cases of confirmed adrenocor-
tical oncocytoma have been associated with metastasis or malignant behavior to the best of our knowledge. It is also important to differentiate adrenocortical oncocytoma from oncocytic adrenocortical carcinoma reported by Hoang et al. [8]. Necrosis, cap- sular extension, and vascular invasion are typically not detected in adrenocortical oncocytoma and these features strongly indicate the diagnosis of adrenocortical oncocytic carcinoma, which may also be exclusively composed of oncocytic adreno- cortical tumor cells. Important consider- ations for surgical pathologists in dealing with adrenocortical oncocytoma are sum- marized as follows:
1. Be well-aware of the entity of adreno- cortical oncocytoma, which is rare but presents important pitfalls.
2. Return a thorough examination to exclude components of clear cells because oncocytoma rarely contains foci of clear cortical tumor cells
3. Document the absence of hemorrhage and/or necrosis, capsular extension, and venous invasion, because adrenocortical oncocytoma is rarely associated with those macroscopic features. If these fea- tures are detected, the diagnosis of oncocytic adrenocortical carcinoma should be made.
4. Inquire as to the absence of adrenocor- tical dysfunction because adrenocortical oncocytomas express no steroidogenic enzymes and generally are considered true non-functioning adrenocortical tumors [7]. This is especially important in distinguishing oncocytomas from androgen-secreting adrenocortical carci- noma because the latter tumors histo- pathologically mimic adrenocortical oncocytoma. Usually these carcinomas contain foci of clear cell components after a thorough sectioning and show increased mitotic activity or necrosis or invasion.
In general one should be very careful in diagnosing malignant adrenocortical oncocytoma.
Pediatric Adrenocortical Carcinoma
Pediatric adrenocortical carcinoma is rare. Its histological features are basically similar to those of adult adrenocortical car- cinoma. However, it is also important to note that the criteria of Weiss [5,6] were based on the analysis of tumors from adult patients from 20 to 70 yr old and no pedi- atric cases were included. When applying the criteria of Weiss to pediatric adreno- cortical carcinoma, the percentage of cases with a good clinical outcome is larger than in adults [9-11], especially in infants. Therefore, pediatric adrenocortical neo- plasm could be overdiagnosed as adrenocor- tical carcinoma. Wieneke et al. proposed the following nine macroscopic and histologi- cal criteria for discerning malignancy in pediatric adrenocortical neoplasms [9]: (1) tumor weight more than 400 g; (2) tumor size more than 10.5 cm; (3) the presence of tumor infiltration into adjacent soft tissues or organs; (4) the presence of tumor invasion into vena cava; (5) the presence of capsular invasion; (6) the pres- ence of venous invasion; (7) the presence of atypical mitosis; (8) the presence of confluent tumor necrosis; and (9) more than 15 mitosis per 20 high power fields. Based on these criteria, the following potential classification was proposed: 1, benign or adenoma when two or fewer above criteria are met; 2, uncertain post- operative clinical behavior when three of the criteria above were met; and 3, malig- nant when four or more of the criteria above are met. However, further investiga- tions such as the study of more cases in different population groups and gene pro- file analyses are needed to establish the criteria of malignancy in pediatric adreno- cortical carcinoma patients.
Immunohistochemical Diagnosis
Recently, molecular and cellular features of human adrenocortical carcinoma have been studied, but not as extensively stud- ied compared with other human malignan- cies [12-14]. The relatively rare frequency of adrenocortical carcinoma prevents investigators from drawing conclusions about the biological significance of results obtained from molecular and cellular stud- ies. In addition, there are no established premalignant conditions in the human adrenal cortex and a transition from adrenocortical adenoma to carcinoma has not been documented. Therefore, the pos- sible significance of molecular and cellular abnormalities detected in individual; or in a small number of carcinoma patients can be very difficult to evaluate or interpret. In addition, human adrenocortical carci- nomas are markedly heterogeneous mor- phologically and functionally even within the same tumor [12]. The number of stro- mal cells including fibroblasts and vascu- lar cells varies from case to case and from area to area in the same case as in other solid human tumors. Therefore, molecu- lar and cellular features of human adreno- cortical carcinoma are clinically of no value and/or significance in differentiation between adrenocortical adenomas and car- cinomas even if meticulously and elegantly performed unless the findings are corre- lated with morphological features. Among these molecular and/or cytological mark- ers that may contribute to the differential diagnosis of adrenocortical carcinoma at least at this juncture, the cell proliferation markers such as Ki-67 and topoisomerase can be of most value [15-20]. Results of recent studies using Ki-67 or MIB1 index for differential diagnosis between adreno- cortical adenoma and carcinoma are sum- marized in Table 1. In the great majority of the cases, if the labeling index (LI) of
| Authors/reference | Cases | Antibody/methods | Results |
|---|---|---|---|
| Iino [15] | A: 28; C: 17 | MIB1 1000 cells at random | A: 0.72 ± 0.12%; C: 6.13 ± 1.64% |
| Martins et al. [17] | Children A: 11; C: 18 | M7240Ab | Children A: 2.5 ± 1.3%; C: 14.4 ± 2.8 % |
| Adults A: 15; C: 6 | 1000 cells at random | Adult A: 0.2± 0.06%; C: 17.8 ±5.8% | |
| Bernini [18] | A (PA 13, NF 13): 26; C: 16 | MIB1 500 cells | PA: 0.53 ± 0.08%; NF: 0.53 ± 0.08%; C: 13.7 ± 3% |
| Wachenfeld [19] | A: 30; C: 10 | MIB1 | A: 2.11 ± 1.01%; C: 11.94 ± 7.58% |
| 500 cells at random | |||
| Terzolo [20] | A: 26; C 11 | MIB1 1000 cells at hot spots | A: 1.13 ± 1.6%; C: 18.58 ± 6.03% |
| Note: ± represents mean ±SD. A, adrenocortical adenoma; C, adrenocortical carcinoma. | |||
Ki-67 is greater than 2.5%, the tumor is most likely adrenocortical carcinoma.
What Pathologists Should Communicate to Clinicians Prior to the Diagnosis of Adrenocortical Malignancy
When dealing with a surgical pathology diagnosis of adrenocortical neoplasms, it is very important to inform clinicians of the following two points.
Never Expect Diagnosis of Adrenocortical Malignancy by Biopsy or Fine-Needle Aspiration in Primary Adrenocortical Neoplasms
Several studies have been reported in fine-needle aspiration or other cytological aspects of adrenocortical neoplasms [21,22]. It is very important to note that adrenocortical carcinoma is associated with marked heterogeneity and an evaluation of overall histological findings is essential for the eventual diagnosis of adrenocortical malignancy. Fine-needle-aspiration cytol- ogy or needle biopsy may provide very important information as to whether the mass could be adrenocortical origin or not but it can by no means provide definitive information to rule it out.
Never Expect to Rule Out Adrenocortical Malignancy by Frozen Sections or Other Intraoperative Consultation in Primary Adrenocortical Neoplasms
This argument is similar to that above. The histological criteria of Weiss are based on histological findings in well-fixed and paraffin-embedded specimens. In addition, as described above, a thorough sectioning or sampling of the specimens is very important in reaching the final diagnosis of adrenocortical malignancy. Therefore, it is important not to attempt to apply the criteria in frozen sections of the resected specimens. However, it is also important to note that intraoperative consultation could provide important information as to whether the tumor is of adrenocortical origin or not.
Determination of Prognostic Factors and Surrogate Markers of Treatment in Adrenocortical Carcinoma
As in malignancies of other organs, it is important to evaluate prognosis of the patients after a diagnosis of adrenocortical carcinoma is established. In addition, recent advances in targeted therapy could result in clinical demand to evaluate
potential surrogate markers in resected specimens of adrenocortical carcinoma, as in the analysis of HER2/neu in deter- mining potential candidates for Herceptin treatment in breast cancer patients.
As expected, complete surgical removal or extirpation resulted in the best chance of survival and clinical stage is still consid- ered the best prognostic factor for patients with adrenocortical carcinoma [23]. Harrison et al. evaluated the pathologic features of prognostic significance for adrenocortical carcinoma after curative resection [24]. They concluded that tumor size, hemorrhage, and mitotic counts cor- related best with survival of patients undergoing curative resection [24]. They further proposed the classification of the patients into low- and high-risk groups based on these three parameters. Stojadi- novic et al. reported that tumor morphol- ogy or histopathology is a better predictor of metastatic risk in patients with adreno- cortical carcinoma than any molecular markers [12]. However, gene expression profiling or gene signatures of individual carcinoma cases may ultimately provide more detailed prediction of clinical out- come in individual patients.
Mitotane or op’DDD is the only adre- nal-specific agent available for the treat- ment of adrenocortical carcinoma [25,26]. Because Mitotane could reverse in vitro multidrug resistance, mitotane has usually been used in combination with various che- motherapeutic agents in advanced cases or in an adjuvant setting following curative surgery of adrenocortical carcinoma [25,26]. Objective response to mitotane is achieved in approx 25% of patients with adrenocortical carcinoma but side effects such as those in GI tract, CNS and respi- ratory system can pose serious problems to the patients [25]. Therefore, it becomes important to select patients for treatment
with mitotane. Currently, exact mecha- nisms of adrenolytic activity of mitotane remain to be elucidated fully but potential surrogate markers could provide important information in the clinical management of the patients. Further investigations are required to identify potential surrogate markers of mitotane in resected specimens of adrenocortical carcinoma.
Differential Diagnosis Between Adrenocortical Primary or Not
In patients who do not manifest any clinical hormonal abnormalities, malignan- cies that must be considered in differential diagnosis of adrenocortical carcinoma at both primary and metastatic sites include renal cell carcinoma, hepatocellular carci- noma, clear cell carcinoma of the ovary and uterus, malignant melanoma, large cell car- cinoma of the lung, and pheochromo- cytoma. Diagnosis of adrenocortical carcinoma is important in these patients because mitotane treatment results in a small but significant increase in mean sur- vival times in the patients with adrenocor- tical carcinoma as described above.
The two most important primary neo- plasms in differential diagnosis of primary adrenocortical carcinoma are renal cell and hepatocellular carcinoma, especially when the lesions are large [27]. In addition, the differential diagnosis between adrenocor- tical carcinoma and metastatic tumor, such as malignant melanoma or large cell carci- noma of the lung or clear cell carcinoma of the ovary or uterus, can be a difficult on occasion [27]. Careful histological exami- nation and detection of markers such as alpha-1 antitrypsin or alpha-fetoprotein or HepPar1 immunoreactivity in hepatocel- lular carcinoma, abundant glycogen or CD10 and RCC Ma immunoreactivity in
A
B
the clear cell types of renal cell carcinoma, S-100 and HMB-45 immunoreactivity in malignant melanoma, and chromogranin or other neuroendocrine markers in pheo- chromocytoma, can all resolve the diagnos- tic ambiguity between adrenocortical carcinoma and other tumors in most cases. However, specific adrenocortical tumor marker can contribute greatly to the dif- ferential diagnosis of these tumors.
The presence of abundant well-devel- oped smooth endoplasmic reticulum and mitochondria with tubular or tubulo- vesicular cristae by electron microscopic observation strongly indicates adrenocor- tical carcinoma. However, these findings are observed only in one half of the cases of adrenocortical carcinoma [2,27]. Dem- onstration of steroidogenic enzymes, espe- cially the enzymes present in only the adrenal cortex, such as P450c11 (11ß- hydroxylase) [28], can be of great help in the diagnosis of adrenocortical carcinoma. However, the patterns of expression of ste- roidogenic enzymes in adrenocortical car- cinoma are different from adrenocortical
adenoma, and some carcinoma cells do not express steroidogenic enzymes involved in corticosteroidogenesis [29,30]. At this juncture, no steroidogenic enzymes can be an immunohistochemical marker specific for adrenocortical carcinoma. Recently, Tartour et al. [31] reported that nuclear immunoreactivity recognized by the D11 monoclonal antibody was highly specific for adrenocortical carcinoma, but immu- noreactivity was only observed in 44% of carcinomas and was restricted to “well-dif- ferentiated types” of adrenocortical carci- noma. Therefore, the practical value of the D11 monoclonal antibody is markedly limited as a marker for adrenocortical carcinoma.
Adrenal 4 binding protein (Ad4BP) or SF-1 is a transcription factor of all steroido- genesis. We have reported that Ad4BP/SF-1 immunoreactivity was demonstrated in almost all the tumor cells of adrenocorti- cal carcinoma including the cells with bizarre nuclei and/or marked cytological atypia, in both histological sections [32] (Fig. 2) and cytology specimens [22],
but not in renal cell carcinoma, hepatocel- lular carcinoma, malignant melanoma, ovarian and uterine clear cell carcinoma, large cell carcinoma of the lung or pheo- chromocytoma. Ad4BP/SF-1 expression is reported in gonadal sex cord-stromal tumors including steroid cell tumor [32,33] but application of Ad4BP/SF-1 immunohistochemistry can nevertheless greatly contribute to the differential diag- nosis of adrenocortical carcinoma from other malignancies both at primary and metastatic sites, even in needle biopsy specimens. In our laboratory, immunostain of Ad4BP/SF-1 has provided superior results, in both specificity and sensitivity, in differential diagnosis between adreno- cortical and non-adrenocortical neoplasms compared to other markers such as inhibin, calretinin, Melan A or others (data not shown). Therefore, immunohisto- or cytochemistry of Ad4BP/SF-1 may be incorporated in the diagnostic laboratories.
References
1. Sasano N. Functional pathology of adreno- cortical endocrine disorders. Trans Soc Pathol Jpn 64:31-54, 1975.
2. Sasano N, Sasano H. The adrenal cortex. In: Kovacs K, Asa SL, eds. Functional endo- crine pathology. Oxford: Blackwell, 1998, 546-584.
3. Gandour MJ, Grizzle WE. A small adreno- cortical carcinoma with aggressive behavior. An evaluation of criteria for malignancy. Arch Pathol Lab Med 110:1076-1079, 1986.
4. Hough AJ, Hollifield JW, Page DL, Hartmann WH. Prognostic factors in adrenal cortex tumors: a mathematical analysis of clinical and morphological data. Am J Clin Pathol 72:390-399, 1979.
5. Weiss LM. Comparable histologic study of 43 metastasizing and nonmetastasizing adreno- cortical tumors. Am J Surg Pathol 8:163-169, 1984.
6. Weiss LM, Medeiros LJ, Vickery AL. Patho- logic features of prognostic significance in
adrenocortical carcinoma. Am J Surg Pathol 13:202-206, 1989.
7. Sasano H, Suzuki T, Sano T, et al. Adrenocor- tical oncocytoma-a true nonfunctioning adrenocortical tumor. Am J Surg Pathol 15:949-956, 1991.
8. Hoang MP, Ayala AG, Albores-Saavedra J. Oncocytic adrenocortical carcinoma: a mor- phologic, immunohistochemical and ultra- structural study of four cases. Mod Pathol 15:973-978, 2002.
9. Wieneke JA, Thompson LDR, Heffess CS. Adrenal cortical neoplasms in the pediatric population. A clinicopathological and immunophenotypic analysis of 83 patients. Am J Surg Pathol 77:867-881, 2003.
10. Lack EE, Mulivihil JJ, Travis WD, et al. Adrenocortical neoplasms in the pediatric and adolescent age group. Clinicopathological study of 30 cases with emphasis on epidemio- logical and prognostic factors. Pathol Annu 27:1-53, 1992.
11. Mickalkiexicz EL, Sandrini R, Figueriredo B, et al. Clinical and outcome characteristics of children with adrenocortical tumors: a report from the international pediatric adrenocorti- cal registry. J Clin Oncol 22:838-845, 2004.
12. Stojadinovic A, Ghossein RA, Hoos A, et al. Adrenocortical carcinoma: clinical, morpho- logic, and molecular characterization. J Clin Oncol 15:941-950, 2002.
13. Stratakis CA. Genetics of adrenocortical tumors: gatekeepers, landscapers and conduc- tors in symphony. Trends Endocrinol Metab 14:404-410, 2003.
14. Sidhu S, Gicquel C, Bambach CP, et al. Clini- cal and molecular aspects of adrenocortical tumorigenesis. ANZ J Surg 73:727-738, 2003.
15. Iino K, Sasano H, Yabuki N, et al. DNA topoisomerase II alpha and Ki-67 in human adrenocortical neoplasms: a possible marker of differentiation between adenomas and car- cinomas. Mod Pathol 10:901-907, 1997.
16. Nakazumi H, Sasano H, Iino K, Ohashi Y, Orikasa S. Expression of cell cycle inhibitor p27 and Ki-67 in human adrenocortical neo- plasms. Mod Pathol 11:1165-1170, 1998.
17. Martins ACP, Cologna AJ, Tucci S, et al. Clini- cal features and immunoexpression of p53, MIB-1 and proliferating cell nuclear antigen in adrenal neoplasms. J Urol 173:2138-2142, 2005.
18. Bernini GP, Moretti A, Viacava P, et al. Apoptosis control and proliferation marker in human normal and neoplastic adrenocortical tissues. Br J Cancer 86:1561-1565, 2002.
19. Wachenfeld C, Beuschlein F, Zwermann O, et al. Discerning malignancy in adrenocorti- cal tumors: are molecular markers useful? Eur J Endocrinol 145:335-341, 2001.
20. Terzolo M, Boccuzzi A, Bovio S, et al. Immunohistochemical assessment of Ki-67 in the differential diagnosis of adrenocortical tumors. Urology 57:176-182, 2001.
21. Krishnamurthy S, Ordonez NG, Shelton TO, et al. Fine-needle aspiration cytology of a case of oncocytic adrenocortical carcinoma. Diagn Cytopathol 22:299-303, 2000.
22. Sasano H, Shizawa S, Nagura H. Adrenocor- tical cytopathology. Am J Clin Pathol 104:161-166, 1995.
23. Ng L, Libertino JM. Adrenocortical carci- noma: diagnosis, evaluation and treatment. J Urol 169:5-11, 2003.
24. Harrison LE, Gaudin PB, Brennan MF. Patho- logic features of prognostic significance for adrenocortical carcinoma after curative resec- tion. Arch Surg 134:181-185, 1999.
25. Hahner S, Fassnacht M. Mitotane for adreno- cortical carcinoma treatment. Curr Opin Investig Drugs 6:386-394, 2005.
26. Berruti A, Terzolo M, Pia A, et al. Mitotane associated with etoposide, doxorubicin, and
cisplatin in treatment of advanced adrenocor- tical carcinoma. Italian group study of adre- nal cancer. Cancer 83:2194-2200, 1998.
27. Neville AM, O’Hare MJ. The human adrenal cortex. Berlin, Germany: Springer-Verlag, 1982.
28. Sasano H, Okamoto M, Sasano N. Immuno- histochemical study of human adrenal cortex with mineralo- and glucocorticoid excess. Virchows Arch [A] 413:313-318, 1988.
29. Sasano H. Localization of steroidogenic enzymes in adrenal cortex and its disorders. Endocrine J 41:471-482, 1994.
30. Sasano H. New approaches in human adreno- cortical pathology: assessment of adrenocor- tical function in surgical specimen of human adrenal glands. Endocr Pathol 3:4-13, 1992.
31. Tartour E, Caillou B, Tenenbaum F, et al. Immunohistochemical study of adrenocorti- cal carcinoma: predictive value of the D11 monoclonal antibody. Cancer 72:3296-3303, 1993.
32. Sasano H, Shizawa S, Suzuki T, et al. Tran- scription factor adrenal 4 binding protein as a marker of adrenocortical malignancy. Hum Pathol 26:1154-1156, 1995.
33. Takayama K, Sasano H, Fukaya T, et al. Immunohistochemical localization of Ad4-bind- ing protein with correlation to steroidogenic enzyme expression in cycling human ovaries and sex cord stromal tumors. J Clin Endocrinol Metab. 80:2815-2821, 1995.