—  SYMPOSIUM #32  —

Molecular Endocrine Pathology
Moderators: Dr. Ricardo Lloyd and Dr. George Kontogeorgos

Section 5 - Adrenal Cortical Tumors

Thomas J. Giordano


Background and Introduction
Adrenal cortical tumors (ACT) are relatively rare. However, these tumors are worthy of attention for several main reasons. First, the incidence of the adrenal "incidentaloma" has risen over the last decade due to improved imaging studies. Second, these tumors are clinically and pathologically fascinating due to their associated hormonal and genetic syndromes. Finally, the therapeutic choices currently available for adrenal cortical carcinoma (ACC) are quite limited. Thus, there is a considerable clinical need for new, more effective and less toxic therapies. Many of these aspects were discussed at a recent international meeting held in Ann Arbor in September 2003 [1].

Histopathology
The majority of ACTs can be adequately evaluated by histopathology. The accurate separation of ACTs into adrenal cortical adenoma (ACA) and carcinoma (ACC) is usually straightforward in most cases using established criteria [2]. Furthermore, valuable prognostic information can also be obtained using objective criteria [3]. Using this prognostic grading system, mitotic activity is assessed by light microscopy and tumors are divided into low- and high-grade groups based on whether tumors have less than or greater than 20 mitoses per 50 high power fields. In my unpublished experience at the University of Michigan, this mitotic activity based grading system is easy to perform and clinically useful.

Immunohistochemistry
Immunohistochemistry (IHC) of ACTs is performed for two reasons. The first reason is to provide support for adrenal cortical differentiation in either a metastatic tumor or a poorly differentiated tumor of the retroperitoneum. In this instance, IHC for α-inhibin, melan-A, and calretinin may be of some utility [4, 5, 6].

The second and more challenging reason to perform IHC on ACTs is to accurately separate ACAs and ACCs. Not surprisingly, most of the work to date has focused on proliferation-related markers such as MIB-1/Ki-67 labelling index or topoisomerase II α [7, 8, 9, 10, 11, 12, 13, 14, 15, 16]. The common theme throughout all of these studies is that ACCs have much higher levels of proliferation that can be exploited diagnostically and prognostically. However, there are still rare ACTs that are difficult to classify with these adjunctive tools and hence occasional tumors still merit a diagnosis of "ACT of uncertain malignant potential".

Genotyping Studies
As with other types of human tumors, ACTs acquire mutations in their DNA that manifest in the typical forms that range from gross chromosomal abnormalities (e.g. rearrangements) to single base substitutions. These mutations have been studied in an effort to better understand the pathogenesis of ACC, but it is also hoped that they can be exploited to develop a robust way to differentiate ACA from ACC.

Comparative Genomic Hybridization
The experimental methods used to examine mutations in ACTs include Comparative Genomic Hybridization (CGH), which is designed to identify large regions of the chromosomes that have either undergone deletion or amplification. By examining a large cohort of ACTs with CGH, it is possible to identify regions that have consistently been altered and thus identify regions that contain either oncogenes or tumor suppressor genes. There have at least been four CHG studies performed on adult ACTs [17, 18, 19, 20]. While it is clear that CGH is a powerful chromosomal discovery tools and the resulting CGH data in ACTs support a model in which ACA can progress to ACC in a stepwise fashion, CGH has not yet yielded a genotype that can be clinically exploited to assist in the diagnosis of these tumors.

Single Marker Genotyping
One of the most successful ways to identify cancer related genes has been to isolate the genes responsible for familial cancer syndromes. There are 2 such syndromes in which ACC is a common manifestation; Beckwith-Wiedemann syndrome (BWS) (Online Mendialian Inheritance in Man (OMIM) entry 130650) and Li-Fraumeni syndrome (OMIM entry 151623). BWS is an overgrowth disorder associated several tumors types. The mutations causing BWS have been tightly linked to the chromosomal region 11p15.5, which is a complex and imprinted region that contains several genes including H19, KIP2, and IGF2. Based on numerous studies, the role of IGF2 in sporadic and familial ACC has been well established [21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33]. IGF2 can undergo a variety of rearrangements, the most common being paternal isodisomy (loss of maternal allele and duplication of paternal allele), that increase the expression of IGF2 and that these are essentially restricted to ACCs.

The Li-Fraumeni syndrome has been linked to mutations of the TP53 gene [34]. Many studies have examined TP53 mutations in ACTs with quite variable results [34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48]. Generally mutations were found in 20 to 67% of ACCs and were rare in ACAs. While the highest mutation frequency reported in ACCs was 67%, this is not high enough to be used clinically to accurately separate benign and malignant tumors. LOH studies hold the greatest diagnostic potential.

Other mutations have been associated with ACTs (reviewed in [49])such as mutations of the MEN1 [50, 51, 52] and PRKAR1A [53, 54, 55, 56], but they have not been demonstrated to have any utility in separating ACAs from ACCs.

Molecular Profiling Studies
One of the difficulties or limitations of using genotyping in a clinical setting is that several different types of mutations can inactivate (or activate) the same gene. Point mutations can be distributed across large genes (i.e. point mutations of MEN1) making their identification technically difficult, and it is also possible to mutate a single gene via distinct mutational mechanisms. For example, the most common type of BRAF mutation in papillary thyroid carcinoma is a point mutation, but less common translocations and amplifications have also been reported [57, 58, 59]. Thus, a method based on DNA sequencing for point mutations will completely fail to identify the other mutations. For this reason, as well as others, there is much excitement over high-throughput methods to examine gene expression in tissues [60, 61, 62, 63, 64]. The development of commercially-available DNA microarrays has permitted their use in a variety of clinicopathologic studies and the first such studies of adrenal cortical tumors have been published.

In a study from our laboratory [28], we used oligonucleotide arrays and small cohort of normal adrenal cortex, ACAs and ACCs identify a gene expression profile that robustly separated benign and malignant tumors, including one low-grade tumor. Using a large group of variably expressed genes selected without reference to pathologic diagnosis, separation of ACA from the other cortical tissues was observed. The one low grade ACC (designated C13) was intermediate in its classification. When a reduced gene list of differentially expressed genes was used, C13 clearly segregated with the other ACCs. This was observed when both Principal component analysis (PCA) and hierarchical clustering was performed. Consistent with the IGF2 molecular work described above, IGF2 expression was greatly increased in the ACCs compared to the other tissues along with many other genes related to increased proliferation. While limited to a small cohort of tumors, this study clearly illustrates the power of molecular profiling approaches for tumor classification and gene discovery.

A second microarray paper used a larger set of tumors but a smaller set of 230 genes [65]. The power of this study comes not from the depth of its array but from their ability to correlate gene expression and patient outcome. Combining these variables, a 22-gene set was developed and shown to possess predictive power.

Collectively, these 2 studies demonstrated clear differences in gene expression between benign and malignant adrenal cortical tumors and illustrate the potential power of molecular profiling approaches based on gene expression for tumor classification. A new study with larger number of tumors is in its early phases at the University of Michigan.

Future Directions
It is difficult to predict the exact path this field with take in the years to come, especially given the rapid evolution of high-throughput technologies to comprehensively investigate various aspects of tumor cell biology. However, it is clear the fields of oncology and pathology are on the edge of a near-radical transformation in which more targeted therapies become available and more intelligent therapeutic choices are made, largely driven by molecular profiling-based assessments of patient's tumors. It will be exhilarating to witness and participate in this transformation as it is applied to the diagnosis, prognosis and treatment of adrenal cortical carcinoma.

References
  1. Schteingart DE, Doherty GM, Gauger PG, et al. Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 2005;12(3):667-80.

  2. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol 1984;8(3):163-9.

  3. Weiss LM, Medeiros LJ, Vickery AL, Jr. Pathologic features of prognostic significance in adrenocortical carcinoma. Am J Surg Pathol 1989;13(3):202-6.

  4. Cho EY, Ahn GH. Immunoexpression of inhibin alpha-subunit in adrenal neoplasms. Appl Immunohistochem Mol Morphol 2001;9(3):222-8.

  5. Jorda M, De MB, Nadji M. Calretinin and inhibin are useful in separating adrenocortical neoplasms from pheochromocytomas. Appl Immunohistochem Mol Morphol 2002;10(1):67-70.

  6. Zhang PJ, Genega EM, Tomaszewski JE, Pasha TL, LiVolsi VA. The role of calretinin, inhibin, melan-A, BCL-2, and C-kit in differentiating adrenal cortical and medullary tumors: an immunohistochemical study. Mod Pathol 2003;16(6):591-7.

  7. Goldblum JR, Shannon R, Kaldjian EP, et al. Immunohistochemical assessment of proliferative activity in adrenocortical neoplasms. Mod Pathol 1993;6(6):663-8.

  8. Sasano H, Imatani A, Shizawa S, Suzuki T, Nagura H. Cell proliferation and apoptosis in normal and pathologic human adrenal. Mod Pathol 1995;8(1):11-7.

  9. Edgren M, Eriksson B, Wilander E, Westlin JE, Nilsson S, Oberg K. Biological characteristics of adrenocortical carcinoma: a study of p53, IGF, EGF-r, Ki-67 and PCNA in 17 adrenocortical carcinomas. Anticancer Res 1997;17(2B):1303-9.

  10. Vargas MP, Vargas HI, Kleiner DE, Merino MJ. Adrenocortical neoplasms: role of prognostic markers MIB-1, P53, and RB. Am J Surg Pathol 1997;21(5):556-62.

  11. 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 carcinomas. Mod Pathol 1997;10(9):901-7.

  12. Nakazumi H, Sasano H, Iino K, Ohashi Y, Orikasa S. Expression of cell cycle inhibitor p27 and Ki-67 in human adrenocortical neoplasms. Mod Pathol 1998;11(12):1165-70.

  13. Gupta D, Shidham V, Holden J, Layfield L. Prognostic value of immunohistochemical expression of topoisomerase alpha II, MIB-1, p53, E-cadherin, retinoblastoma gene protein product, and HER-2/neu in adrenal and extra-adrenal pheochromocytomas. Appl Immunohistochem Mol Morphol 2000;8(4):267-74.

  14. Terzolo M, Boccuzzi A, Bovio S, et al. Immunohistochemical assessment of Ki-67 in the differential diagnosis of adrenocortical tumors. Urology 2001;57(1):176-82.

  15. Arola J, Salmenkivi K, Liu J, Kahri AI, Heikkila P. p53 and Ki67 in adrenocortical tumors. Endocr Res 2000;26(4):861-5.

  16. Takehara K, Sakai H, Shono T, Irie J, Kanetake H. Proliferative activity and genetic changes in adrenal cortical tumors examined by flow cytometry, fluorescence in situ hybridization and immunohistochemistry. Int J Urol 2005;12(2):121-7.

  17. Dohna M, Reincke M, Mincheva A, Allolio B, Solinas-Toldo S, Lichter P. Adrenocortical carcinoma is characterized by a high frequency of chromosomal gains and high-level amplifications. Genes Chromosomes Cancer 2000;28(2):145-52.

  18. Kjellman M, Kallioniemi OP, Karhu R, et al. Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy. Cancer Res 1996;56(18):4219-23.

  19. Sidhu S, Marsh DJ, Theodosopoulos G, et al. Comparative genomic hybridization analysis of adrenocortical tumors. J Clin Endocrinol Metab 2002;87(7):3467-74.

  20. Zhao J, Roth J, Bode-Lesniewska B, Pfaltz M, Heitz PU, Komminoth P. Combined comparative genomic hybridization and genomic microarray for detection of gene amplifications in pulmonary artery intimal sarcomas and adrenocortical tumors. Genes Chromosomes Cancer 2002;34(1):48-57.

  21. Boulle N, Logie A, Gicquel C, Perin L, Le Bouc Y. Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors. J Clin Endocrinol Metab 1998;83(5):1713-20.

  22. Erickson LA, Jin L, Sebo TJ, et al. Pathologic features and expression of insulin-like growth factor-2 in adrenocortical neoplasms. Endocr Pathol 2001;12(4):429-35.

  23. Fottner C, Hoeflich A, Wolf E, Weber MM. Role of the insulin-like growth factor system in adrenocortical growth control and carcinogenesis. Horm Metab Res 2004;36(6):397-405.

  24. Gao ZH, Suppola S, Liu J, Heikkila P, Janne J, Voutilainen R. Association of H19 promoter methylation with the expression of H19 and IGF-II genes in adrenocortical tumors. J Clin Endocrinol Metab 2002;87(3):1170-6.

  25. Gicquel C, Bertagna X, Gaston V, et al. Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001;61(18):6762-7.

  26. Gicquel C, Bertagna X, Schneid H, et al. Rearrangements at the 11p15 locus and overexpression of insulin-like growth factor-II gene in sporadic adrenocortical tumors. J Clin Endocrinol Metab 1994;78(6):1444-53.

  27. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: study on a series of 82 tumors. J Clin Endocrinol Metab 1997;82(8):2559-65.

  28. Giordano TJ, Thomas DG, Kuick R, et al. Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003;162(2):521-31.

  29. Ilvesmaki V, Kahri AI, Miettinen PJ, Voutilainen R. Insulin-like growth factors (IGFs) and their receptors in adrenal tumors: high IGF-II expression in functional adrenocortical carcinomas. J Clin Endocrinol Metab 1993;77(3):852-8.

  30. Liu J, Kahri AI, Heikkila P, Ilvesmaki V, Voutilainen R. H19 and insulin-like growth factor-II gene expression in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 1995;80(2):492-6.

  31. Liu J, Kahri AI, Heikkila P, Voutilainen R. Ribonucleic acid expression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 1997;82(6):1766-71.

  32. Logie A, Boulle N, Gaston V, et al. Autocrine role of IGF-II in proliferation of human adrenocortical carcinoma NCI H295R cell line. J Mol Endocrinol 1999;23(1):23-32.

  33. Weber MM, Fottner C, Wolf E. The role of the insulin-like growth factor system in adrenocortical tumourigenesis. Eur J Clin Invest 2000;30 Suppl 3:69-75.

  34. Sameshima Y, Tsunematsu Y, Watanabe S, et al. Detection of novel germ-line p53 mutations in diverse-cancer-prone families identified by selecting patients with childhood adrenocortical carcinoma. J Natl Cancer Inst 1992;84(9):703-7.

  35. Barzon L, Chilosi M, Fallo F, et al. Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol 2001;145(2):207-12.

  36. Kobayashi H, Usui T, Fukata J, Yoshimasa T, Oki Y, Nakao K. Mutation analysis of Gsalpha, adrenocorticotropin receptor and p53 genes in Japanese patients with adrenocortical neoplasms: including a case of Gsalpha mutation. Endocr J 2000;47(4):461-6.

  37. Latronico AC, Pinto EM, Domenice S, et al. An inherited mutation outside the highly conserved DNA-binding domain of the p53 tumor suppressor protein in children and adults with sporadic adrenocortical tumors. J Clin Endocrinol Metab 2001;86(10):4970-3.

  38. Lin SR, Lee YJ, Tsai JH. Mutations of the p53 gene in human functional adrenal neoplasms. J Clin Endocrinol Metab 1994;78(2):483-91.

  39. Ohgaki H, Kleihues P, Heitz PU. p53 mutations in sporadic adrenocortical tumors. Int J Cancer 1993;54(3):408-10.

  40. Olivier M, Goldgar DE, Sodha N, et al. Li-Fraumeni and related syndromes: correlation between tumor type, family structure, and TP53 genotype. Cancer Res 2003;63(20):6643-50.

  41. Pinto EM, Billerbeck AE, Fragoso MC, Mendonca BB, Latronico AC. Deletion mapping of chromosome 17 in benign and malignant adrenocortical tumors associated with the Arg337His mutation of the p53 tumor suppressor protein. J Clin Endocrinol Metab 2005;90(5):2976-81.

  42. Pinto EM, Billerbeck AE, Villares MC, Domenice S, Mendonca BB, Latronico AC. Founder effect for the highly prevalent R337H mutation of tumor suppressor p53 in Brazilian patients with adrenocortical tumors. Arq Bras Endocrinol Metabol 2004;48(5):647-50.

  43. Reincke M, Karl M, Travis WH, et al. p53 mutations in human adrenocortical neoplasms: immunohistochemical and molecular studies. J Clin Endocrinol Metab 1994;78(3):790-4.

  44. Reincke M, Wachenfeld C, Mora P, et al. p53 mutations in adrenal tumors: Caucasian patients do not show the exon 4 "hot spot" found in Taiwan. J Clin Endocrinol Metab 1996;81(10):3636-8.

  45. Sandrini F, Villani DP, Tucci S, Moreira AC, de Castro M, Elias LL. Inheritance of R337H p53 gene mutation in children with sporadic adrenocortical tumor. Horm Metab Res 2005;37(4):231-5.

  46. Sidhu S, Martin E, Gicquel C, et al. Mutation and methylation analysis of TP53 in adrenal carcinogenesis. Eur J Surg Oncol 2005;31(5):549-54.

  47. Varley JM, McGown G, Thorncroft M, et al. Are there low-penetrance TP53 Alleles? evidence from childhood adrenocortical tumors. Am J Hum Genet 1999;65(4):995-1006.

  48. Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA, Malkin D. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst 1994;86(22):1707-10.

  49. Libe R, Bertherat J. Molecular genetics of adrenocortical tumours, from familial to sporadic diseases. Eur J Endocrinol 2005;153(4):477-87.

  50. Heppner C, Reincke M, Agarwal SK, et al. MEN1 gene analysis in sporadic adrenocortical neoplasms. J Clin Endocrinol Metab 1999;84(1):216-9.

  51. Schulte KM, Heinze M, Mengel M, et al. MEN I gene mutations in sporadic adrenal adenomas. Hum Genet 1999;105(6):603-10.

  52. Schulte KM, Mengel M, Heinze M, et al. Complete sequencing and messenger ribonucleic acid expression analysis of the MEN I gene in adrenal cancer. J Clin Endocrinol Metab 2000;85(1):441-8.

  53. Bertherat J, Groussin L, Sandrini F, et al. Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity. Cancer Res 2003;63(17):5308-19.

  54. Bossis I, Voutetakis A, Bei T, Sandrini F, Griffin KJ, Stratakis CA. Protein kinase A and its role in human neoplasia: the Carney complex paradigm. Endocr Relat Cancer 2004;11(2):265-80.

  55. Groussin L, Jullian E, Perlemoine K, et al. Mutations of the PRKAR1A gene in Cushing's syndrome due to sporadic primary pigmented nodular adrenocortical disease. J Clin Endocrinol Metab 2002;87(9):4324-9.

  56. Libe R, Mantovani G, Bondioni S, et al. Mutational analysis of PRKAR1A and Gs(alpha) in sporadic adrenocortical tumors. Exp Clin Endocrinol Diabetes 2005;113(5):248-51.

  57. Ciampi R, Nikiforov YE. Alterations of the BRAF Gene in Thyroid Tumors. Endocr Pathol 2005;16(3):163-72.

  58. Ciampi R, Zhu Z, Nikiforov YE. BRAF copy number gains in thyroid tumors detected by fluorescence in situ hybridization. Endocr Pathol 2005;16(2):99-105.

  59. Ciampi R, Knauf JA, Kerler R, et al. Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer. J Clin Invest 2005;115(1):94-101.

  60. Lakhani SR, Ashworth A. Microarray and histopathological analysis of tumours: the future and the past? Nat Rev Cancer 2001;1(2):151-7.

  61. Mischel PS, Cloughesy TF, Nelson SF. DNA-microarray analysis of brain cancer: molecular classification for therapy. Nat Rev Neurosci 2004;5(10):782-92.

  62. Snijders AM, Meijer GA, Brakenhoff RH, van den Brule AJ, van Diest PJ. Microarray techniques in pathology: tool or toy? Mol Pathol 2000;53(6):289-94.

  63. Alizadeh AA, Ross DT, Perou CM, van de Rijn M. Towards a novel classification of human malignancies based on gene expression patterns. J Pathol 2001;195(1):41-52.

  64. Giordano TJ. Gene expression profiling of endocrine tumors using DNA microarrays: progress and promise. Endocr Pathol 2003;14(2):107-16.

  65. de Fraipont F, El Atifi M, Cherradi N, et al. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005;90(3):1819-29.