Investigation of BRAF and CTNNB1 activating mutations in adrenocortical tumors
G. Masi1, E. Lavezzo1, M. Iacobone2, G. Favia2, G. Palù1*, and L. Barzon1*
1Department of Histology, Microbiology, and Medical Biotechnologies; 2Department of Surgical and Gastroenterological Sciences, University of Padua, Padua, Italy
ABSTRACT. Background: Activating mutations of the BRAF oncogene play a central role in the development of various cancer types, but their role in human adrenocortical tumors is unknown. At variance, activating mutations of another onco- gene, CTNNB1, which encodes ß-catenin, have been shown to be common events in both benign and malignant adreno- cortical tumors. Aim: To investigate the prevalence of BRAF and CTNNB1 activating mutations in sporadic adrenocortical tumors. Materials and methods: Tissue samples from 15 adrenocortical carcinomas and 41 adrenocortical adenomas were investigated for the presence of BRAF and CTNNB1 ac-
tivating mutations by PCR amplification and direct sequenc- ing. Results: An advanced invasive non-functioning adreno- cortical carcinoma carried a somatic heterozygous BRAF V600E mutation, while 4 functioning and 4 non-functioning adenomas and 3 functioning carcinomas carried different CTNNB1 activating mutations. Conclusions: Activating BRAF somatic mutations may be occasionally found in advanced adrenocortical carcinomas, while CTNNB1 activating muta- tions are early and common events in adrenal tumorigenesis. (J. Endocrinol. Invest. 32: 597-600, 2009) @2009, Editrice Kurtis
INTRODUCTION
Sporadic adenomas/hyperplasia of the adrenal cortex are frequent incidental findings in the general population, whereas adrenocortical carcinomas are very rare and as- sociated with a poor prognosis (1). The molecular patho- genesis of adrenocortical cancer is not yet completely understood, even though recent advances have high- lighted the involvement of both tumor suppressor genes, such as TP53, MEN1, PRKAR1A, and oncogenes, such as IGF2, CTNNB1, and RAS (2).
Among oncogenes, activating mutations of the CTNNB1 gene and deregulation of the encoded ß-catenin protein appear to be early and common events in adrenal tu- morigenesis. In fact, CTNNB1 somatic mutations have been detected in both benign and malignant adreno- cortical tumors, with a prevalence of 27% and 31%, re- spectively (3). The relatively high prevalence of activat- ing CTNNB1 mutations in benign adrenocortical tumors, including primary pigmented nodular adrenocortical dis- eases associated with PRKAR1A mutations, has been con- firmed in other studies (4, 5). Like in other types of tu- mors, activating mutations mostly affect residues in CTNNB1 exon 3 that are crucial for ß-catenin targeted degradation, and result in cytoplasmic and nuclear ac- cumulation with subsequent increase in ß-catenin tran- scriptional activity (3).
Ras proteins are membrane-associated proteins that are frequently mutated in human malignancies, but their role
in development of adrenal tumors is still controversial. In fact, somatic mutations of K-ras have been reported in 46% of Conn’s adenomas (6), but other studies have not found any Ras mutation (7, 8). The B-RAF kinase is a downstream effector of the RAS protein and part of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signal transduction pathway. Activating mutations of the BRAF oncogene, generally represented by the amino acid substitution V600E, are key events in the development of numerous cancer types, such as melanoma, thyroid, colon, ovarian, and stomach cancer (9, 10), which are also linked to Wnt/B-catenin pathway activation (11). The involvement of BRAF muta- tions in the development of human adrenocortical tu- mors has not been extensively investigated so far. Davies et al. (9) tested for BRAF mutations the adrenocortical carcinoma cell lines NCI-H295 and SW13 and did not de- tect any genetic abnormalities. Regarding adrenal medullary tumors, two reports investigated the presence of BRAF-activating mutations in a total of 69 pheochro- mocytomas, but did not find any mutation (12, 13).
Aim of this study was to investigate a series of function- ing and non-functioning, benign and malignant adreno- cortical tumors for the presence of mutations in BRAF ex- on 15 and in CTNNB1 exon 3, which are the exons where most activating mutations are detected.
MATERIALS AND METHODS Patients and tissue samples
The presence of somatic BRAF and CTNNB1 mutations was screened in tissue samples from 15 adrenocortical carcinomas, 11 cortisol-producing adenomas, 19 aldosterone-producing adenomas, 1 testosterone-producing adenoma, and 10 non- functioning adrenocortical adenomas. Tissue samples were ob- tained from a series of 56 consecutive patients (Table 1) who underwent adrenalectomy at the Endocrine Surgery Unit of the University of Padua in the period between January 2005 and May 2007. Peripheral whole blood samples were also available
*G. Palù and L. Barzon share senior authorship.
Key-words: Adrenocortical carcinoma, adrenocortical neoplasm, ß-catenin, BRAF, mutation analysis, oncogene.
Correspondence: L. Barzon, MD, and G. Palù, MD, Department of Histology, Micro- biology, and Medical Biotechnologies, University of Padova, Via A. Gabelli 63, 1-35128 Padova, Italy.
E-mail: luisa.barzon@unipd.it; giorgio.palu@unipd.it Accepted February 25, 2009.
First published online May 15, 2009.
| Parameter | Value |
|---|---|
| Adrenocortical adenomas | 41 |
| Female/male | 25/16 |
| Median age (range) | 52 yr (21-79 yr) |
| Function | |
| Cortisol-producing | 11 |
| Aldosterone-producing | 19 |
| Testosterone-producing | 1 |
| Non-fuctioning | 10 |
| Adrenocortical carcinomas | 15 |
| Female/male | 7/8 |
| Median age (range) | 53 yr (37-73 yr) |
| Function | |
| Cortisol-producing | 4 |
| Cortisol- and androgen-producing | 4 |
| Aldosterone-producing | 1 |
| Non-fuctioning | 6 |
| Median tumor size (range) | 140 mm (58-180 mm) |
| Tumor stage | |
| II | 4 |
| III | 3 |
| IV | 8 |
from all patients for genetic testing. All patients gave their in- formed consent to participate in the study, which was approved by the local Ethics Committee. Diagnosis of malignancy was performed according to the histopathologic criteria proposed by Weiss et al. (14), with modification proposed by Aubert et al. (15). Briefly, an adrenocortical neoplasm was defined as malig- nant in the presence of 3 or more of the following criteria: a) high nuclear grade, according to the Fuhrman criteria (16), b) more than 5 mitoses per 50 high-power fields, c) atypical mi- totic figures, d) less than 25% of tumor cells are clear cells, e) diffuse architecture (>33% of tumor), f) necrosis, g) venous in- vasion, h) sinusoidal invasion, and i) capsular invasion. Tissue samples were taken from central non-necrotic areas of tumor masses. Microscopic examination of samples estimated that over 80% of cells were tumor cells.
DNA purification and detection of BRAF and CTNNB1 activating mutations
Genomic DNA was isolated from whole blood samples and frozen tissues using QIAmp DNA Mini Kit (Qiagen GmbH,
Hilden, Germany). BRAF exon 15 was amplified by PCR using oligonucleotide primer sequences reported by Davies et al. (9), as previously reported (17). CTNNB1 exon 3 and flanking in- tronic sequences were amplified with the primer pair used by Tissier et al. (3). Bidirectional sequencing of PCR products was performed by using an ABI PRISM BigDye terminators v3.1 cy- cle sequencing kit (Applied Biosystems, Foster City, CA, USA); sequences were run on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and compared with the Consen- sus Coding Sequences CCDS 5863.1 (BRAF) and CCDS 2694.1 (CTNNB1). The presence of mutations was confirmed by re- peating PCR and sequencing on newly purified DNA samples.
RESULTS
Out of 56 adrenocortical tumors screened for the pres- ence of BRAF mutations, only an advanced non-func- tioning adrenocortical carcinoma carried a somatic T1799A transversion, resulting in the common V600E mu- tation. Pathological examination of the tumor showed a poorly differentiated adrenocortical carcinoma, 12 cm in diameter and infiltrating the inferior vena cava. The tu- mor was composed of fasciculata-like cells, with high mi- totic index, atypical mitotic figures, nuclear atypia, dif- fuse architecture, extensive regressive changes, venous and capsular invasion, with involvement of adjacent soft tissues and lymph node metastases.
Somatic mutations in CTNNB1 were detected in 3 out 15 (20%) adrenocortical carcinomas and in 8 out of 41 (20%) adrenocortical adenomas (Table 2). Adenomas, either functioning and non-functioning, were character- ized by the presence of mutations involving Ser45, whereas carcinomas carried mutations affecting amino acids Asp32, His36, and Thr41. Two adrenocortical ade- nomas carried two point mutations affecting amino acids at both positions 44 and 45, while an adenoma carried an in-frame deletion of 33 nucleotides leading to the loss of 11 aminoacids (p.Ala43_Glu53del). The adreno- cortical carcinoma with BRAF mutation had no additional mutations in CTNNB1. No BRAF and CTNNB1 mutations were detected in DNA purified from peripheral blood of patients.
| Patient sex/age (yr) | Clinical syndrome | Urinary cortisol* (nmol/24h) | Aldosterone/ PRA ratio ** | Histological diagnosis | Tumor side/size (mm) | McFarlane staging | CTNNB1 mutation |
|---|---|---|---|---|---|---|---|
| M/43 | Subclinical CS | 420 | 25 | Adenoma | Left/30 | I | Ser45Pro |
| F/55 | CS | 2150 | 37 | Adenoma | Right/30 | I | Ser45Pro |
| M/69 | Hyperaldosteronism | 310 | 180 | Adenoma | Left/10 | I | Ser45Pro |
| F/66 | Hyperaldosteronism | 268 | 248 | Adenoma | Right/15 | I | Ala43_Glu53del |
| M/79 | Nonfunctioning | 254 | 18 | Adenoma | Right/45 | I | Ser45Pro |
| M/54 | Nonfunctioning | 180 | 10 | Adenoma | Left/48 | I | Ser45Phe |
| M/62 | Nonfunctioning | 275 | 28 | Adenoma | Right/50 | I | Pro44Ala+Ser45Pro |
| M/72 | Nonfunctioning | 140 | 14 | Adenoma | Left/45 | I | Pro44Ala+Ser45Pro |
| F/55 | Virilization + CS | 1870 | 30 | Carcinoma | Right/150 | IV | His36Pro |
| F/47 | Subclinical CS | 530 | 22 | Carcinoma | Right/60 | IV | Thr41Pro |
| M/52 | Hyperaldosteronism | 320 | 220 | Carcinoma | Right/160 | IV | Asp32His |
CS: Cushing’s syndrome; PRA: plasma renin activity; M: male; F: female. *: urinary cortisol value represents the mean of 3 measurement of 24-h urinary free cortisol (normal values, 82-330 nmol/24h); **: aldosterone/PRA ratio was measured as the ratio between upright plasma aldosterone (ng per dl) and PRA (ng per ml per h). Details on laboratory methods for hormone measurements have been previously reported (18).
DISCUSSION
In this study, which examined the mutation status of BRAF exon 15 in a series of 56 adrenocortical tumors, BRAF-activating mutation was only detected in an ad- vanced adrenocortical carcinoma, indicating the in- volvement of this oncogene in the development of adrenocortical tumors is only occasional. While review- ing our manuscript, another study on BRAF mutation analysis in adrenocortical carcinomas was published (19). Also this study demonstrated that the prevalence of BRAF activating mutation is low in adrenocortical carci- nomas, being detected only in 2 out of 35 cases (19).
The V600E mutation, which is the most commonly de- tected mutation in many types of tumors, such as melanoma, papillary thyroid cancer, and colon cancer, results in a 500-fold increase in kinase activity, thus in- ducing constitutive ERK and nuclear factor K-B constitu- tive signaling in response to this hyperactivation (20). BRAF V600E has been demonstrated to induce senes- cence in benign tumors of melanocytes (21, 22), but it activates cell growth and proliferation in cancer (20). Functional studies are needed to characterize the effect of this mutation in adrenocortical cells. In our study, the adrenocortical cancer carrying the somatic BRAF muta- tion was very aggressive and highly proliferating, and could have accumulated several other genetic abnor- malities besides BRAF mutation. However, it is possible that the BRAF mutation significantly contributed to the aggressive phenotype of this cancer.
The rarity of the occurrence of BRAF alterations in the development of endocrine neoplasms other than papil- lary thyroid carcinomas, in which the average prevalence of BRAF mutations is 44% (23), was previously reported by Perren et al. (12), who investigated a total of 130 en- docrine tumors and found mutations only in papillary thy- roid carcinomas and in a single case of malignant gastric carcinoid with liver metastasis. Likewise, a study on pitu- itary adenomas showed that only a non-functioning pi- tuitary adenoma out of 50 investigated cases carried the V600E activating mutation (24).
With regard to ß-catenin, our study confirms that somatic activating mutations of CTNNB1 are frequent in adrenal cortex tumorigenesis, and are found with equal preva- lence (20%) in both benign and malignant adrenocorti- cal tumors, as previously reported by Tissier et al. (3). Moreover, in agreement with this study (3), we observed that CTNNB1-activating mutations were relatively more frequent in non-functioning (4 out of 10, 40%) than in functioning (4 out of 31, 13%) adrenocortical adenomas. Data in the literature are however conflicting. In fact, Tadjine et al. (4) identified CTNNB1 mutations more fre- quently in functioning than in non-functioning adreno- cortical adenomas. So, investigation of CTNNB1 muta- tions in larger series of adrenocortical tumors is needed to clarify whether abnormal ß-catenin activation is asso- ciated with alterations of steroidogenesis or with sever- ity of malignant disease. In this regard, in the mouse model, ß-catenin has been recently demonstrated to play a crucial role in adrenocortical development and in maintenance of the adult organ (25). In adrenocortical cells, ß-catenin synergizes with steroidogenic factor 1 to regulate the expression of a subset of target genes, such
those encoding dax-1, inhibin-a, steroidogenic enzymes, etc., whose expression is essential to stimulate adreno- cortical proliferation and steroidogenic function (25). In the adult adrenal cortex, ß-catenin expressing cells are restricted to the subcapsular area of the cortex and might represent the adrenocortical niche/stem-progen- itor unit (25).
In conclusion, our study confirms the central role of CTNNB1 somatic defects in adrenocortical tumorigene- sis, and demonstrates the occasional presence of an ac- tivating BRAF mutation in an adrenocortical carcinoma. Although rare, detection of activating BRAF mutations in adrenocortical carcinomas may allow identifying candi- date patients for tailored therapy with B-RAF kinase in- hibitors.
ACKNOWLEDGMENTS
This work was supported by ex-60% funds from University of Padova to L. Barzon.
REFERENCES
1. Barzon L, Sonino N, Fallo F, Palù G, Boscaro M. Prevalence and natural history of adrenal incidentalomas. Eur J Endocrinol 2003, 149: 273-85.
2. Libè R, Fratticci A, Bertherat J. Adrenocortical cancer: pathophys- iology and clinical management. Endocr Relat Cancer 2007, 14: 13-28.
3. Tissier F, Cavard C, Groussin L, et al. Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tu- mors. Cancer Res 2005, 65: 7622-7.
4. Tadjine M, Lampron A, Ouadi L, Bourdeau I. Frequent mutations of beta-catenin gene in sporadic secreting adrenocortical adenomas. Clin Endocrinol (Oxf) 2008, 68: 264-70.
5. Gaujoux S, Tissier F, Groussin L, et al. Wnt/beta-catenin and 3’,5’- cyclic adenosine 5’-monophosphate/protein kinase A signaling pathways alterations and somatic beta-catenin gene mutations in the progression of adrenocortical tumors. J Clin Endocrinol Metab 2008, 93: 4135-40.
6. Lin SR, Tsai JH, Yang YC, Lee SC. Mutations of K-ras oncogene in human adrenal tumours in Taiwan. Br J Cancer 1998, 77: 1060-5.
7. Moul JW, Bishoff JT, Theune SM, Chang EH. Absent ras gene mu- tations in human adrenal cortical neoplasms and pheochromocy- tomas. J Urol 1993, 149: 1389-94.
8. Ocker M, Sachse R, Rico A, Hensen J. PCR-SSCP analysis of hu- man adrenocortical adenomas: absence of K-ras gene mutations. Exp Clin Endocrinol Diabetes 2000, 108: 513-4.
9. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002, 417: 949-54.
10. . Michaloglou C, Vredeveld LC, Mooi WJ, Peeper DS. BRAF (E600) in benign and malignant human tumours. Oncogene 2008, 27: 877-95.
11. Polakis P. Wnt signaling and cancer. Genes Dev 2000, 14: 1837-51.
12. Perren A, Schmid S, Locher T, et al. BRAF and endocrine tumors: mutations are frequent in papillary thyroid carcinomas, rare in en- docrine tumors of the gastrointestinal tract and not detected in other endocrine tumors. Endocr Relat Cancer 2004, 11: 855-60.
13. Geli J, Kiss N, Karimi M, et al. Global and regional CpG methy- lation in pheochromocytomas and abdominal paragangliomas: association to malignant behavior. Clin Cancer Res 2008, 14: 2551-9.
4. Weiss LM, Medeiros LJ, Vickery AL Jr. Pathologic features of prog- nostic significance in adrenocortical carcinoma. Am J Surg Pathol 1989, 13: 202-6.
15. Aubert S, Wacrenier A, Leroy X. Weiss system revisited: a clinico- pathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol 2002, 26: 1612-9.
16. Greene LF, Page DL, Fritz A, Batch M, Haller DG, Morrow M. American Joint Committee on Cancer (AJCC) Cancer staging man- ual, 6th edn. New York: Springer 2002.
17. Barzon L, Masi G, Boschin IM, et al. Characterization of a novel complex BRAF mutation in a follicular variant papillary thyroid car- cinoma. Eur J Endocrinol 2008, 159: 77-80.
18. Barzon L, Fallo F, Sonino N, Boscaro M. Development of overt Cushing’s syndrome in patients with adrenal incidentaloma. Eur J Endocrinol 2002, 146: 61-6.
19. Kotoula V, Sozopoulos E, Litsiou H, et al. Mutational analysis of the BRAF, RAS and EGFR genes in human adrenocortical carcinomas. Endocr Relat Cancer 2009, 16: 565-72.
20. Dhomen N, Marais R. New insight into BRAF mutations in cancer. Curr Opin Genet Dev 2007, 17: 31-9.
21. Michaloglou C, Vredeveld LC, Soengas MS, et al. BRAF E600-as- sociated senescence-like cell cycle arrest of human naevi. Nature 2005, 436: 720-4.
22. Gray-Schopfer VC, Cheong SC, Chong H, et al. Cellular senes- cence in naevi and immortalisation in melanoma: a role for p16? Br J Cancer 2006, 95: 496-505.
23. Xing M. BRAF mutation in papillary thyroid cancer: pathogenic role, molecular bases, and clinical implications. Endocr Rev 2007, 28: 742-62.
24. De Martino I, Fedele M, Palmieri D, et al. B-RAF mutations are a rare event in pituitary adenomas. J Endocrinol Invest 2007, 30: RC1-3.
25. Kim AC, Reuter AL, Zubair M, et al. Targeted disruption of B- catenin in Sf1-expressing cells impairs development and mainte- nance of the adrenal cortex. Development 2008, 135: 2593-602.