JEI_08_157_Ruggeri.qxp:Layout 1 13-01-2009 9:26 Pagina 946

J. Endocrinol. Invest. 31: 946-949, 2008

RAPID COMMUNICATION Non-functioning pituitary adenomas infrequently harbor G-protein gene mutations R.M. Ruggeri1, L. Santarpia1,2, L. Curtò1, M.L. Torre1, M. Galatioto3, S. Galatioto3, F. Trimarchi1, and S. Cannavò1 1Department of Medicine and Pharmacology, Section of Endocrinology, Univerity of Messina, Messina, Italy; 2Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA; 3Department of Pathology, University of Messina, Messina, Italy

ABSTRACT. Background: Mutations of the genes encoding the α subunit of the stimulatory G protein (Gs) and of the inhibiting Gi2 protein (GNAS1 and GNAI2 genes, respectively) have been described in various endocrine neoplasias, including pituitary tumors. Aim: To search for mutations of GNAS1 and GNAI2 in a continuous series of non-functioning pituitary adenoma (NF- PA) patients neurosurgically treated. Subjects and methods: The surgical samples of 22 patients who have been defined and char- acterized on a clinical, biochemical, histological, and immunohistochemical point of view have been processed for investigating the presence of the above mutations by PCR amplification of the hot spots exons 8 and 9 of GNAS1, and exons 5 and 6 of GNAI2, followed by direct sequencing. Moreover, the promoter region of GNAI2, in order to assess the prevalence of single nu- cleotide polymorphisms (SNP), was investigated in the same series. Results: A CGT>TGT mutation at codon 201 of GNAS1 gene in a single case of NFPA was found, but no mutation of GNAI2A was demonstrated. Conclusions: This finding suggests and con- firms that G-protein mutations are rare and not crucial in NFPA development. Additionally, we found a silent SNP at codon 318 in the promoter of the Gi2α gene in one out of the 22 NFPA. (J. Endocrinol. Invest. 31: 946-949, 2008) ©2008, Editrice Kurtis

INTRODUCTION In addition to GNAS1, also the gene encoding the α subunit of Non-functioning pituitary adenomas (NFPA) occur at a rela- the Gi2 protein (the gip oncogene or GNAI2A), mapped to chro- tively high frequency, accounting for about 20-30% of all ante- mosome 3p21, has been screened for mutations in NFPA (5). rior pituitary tumors in adults (1), as well as in a large unpub- GNAI2A mutations have been identified in a small proportion of lished series of 309 pituitary adenomas referred to our Unit. NFPA (9, 10) as well as in one corticotroph adenoma (12), but Since it has been demonstrated that, like the other pituitary in none of the GH and PRL-secreting adenomas studied so far (9, tumor types, NFPA are monoclonal in origin (2-3), search for 14). Whether or not mutated Gi proteins may activate mitogenic gene mutations or rearrangements involved in tumor growth signals in pituitary cells remains to be understood (5). have been performed, but it has been largely unfruitful (1, 4, 5). In the present study, we have examined 22 NFPA with the The results obtained in the recent years showing a quite high purpose to search for mutations in the GNAS1 and GNAI2 prevalence of mutations of the gsp oncogene (or GNAS1) in genes. Moreover, we analyzed the possible association be- GH-secreting adenomas created an expectancy for a similar tween the C318G single nucleotide polymorphism (SNP) of ©prevalence in other types of pituitary tumors (6-8). the GNAI2 promoter region and pituitary tumorigenesis. The gsp2008,oncogene (or GNAS1 gene), loca Editricelized on chromosome Kurtis 20q13, encodes the α subunit of the stimulatory G protein (Gs)(6). Mutations of the GNAS1 lead to the constitutive activa- MATERIALS AND METHODS tion of the Gsα suFORbunit by inhibitin PERSONALg its intrinsic GTPase activity, Patients USE and tissues’ ONLY collection and result in an increased cAMP production and subsequent over- Tumor samples were obtained from 22 consecutive patients activation of specific pathways (i.e. cAMP-PKA-CREB pathways) referred to our University Hospital where have been evaluat- involved in both cell growth and specific programmes of cell dif- ed and underwent neurosurgery. ferentiation. These mutations are found in about 40% of GH-se- Six patients were females (median age 47 yr, range 26-67) and 16 creting adenomas (7, 8). Conversely, GNAS1 mutations have males (median age 58 yr, range 41-75). The functional classification been demonstrated only in a minority of the NFPA analyzed (9- of the pituitary adenomas was based on the currently accepted 11), as well as in a small number of ACTH-secreting adenomas clinical and hormonal criteria. No patient in this group had any (12, 13), and their real pathogenetic role in the development of clinical or biochemical evidence of excessive hormonal secretion. pituitary adenomas other than GH-omas remain uncertain. As summarized in Table 1, the tumor size, evaluated by magnet- ic resonance imaging, ranged from 14 to 40 mm in diameter. Cav- ernous sinus invasion was revealed in 11/22 patients, while bone erosions were evident in 13 patients at diagnosis. During a 5-yr- R.M. Ruggeri and L. Santarpia equally contributed to this work. long follow-up after surgery, the disease recurred in 6 patients. Key-words: 318C>G single nucleotide polymorphism, GNAI2A, GNAS1, G-proteins, non- The hospital Ethics Committee approved the study protocol. functioning pituitary adenomas. Correspondence: R.M. Ruggeri, MD, Sezione di Endocrinologia, Dipartimento Clinico-Sper- Histological and immunohistochemical evaluation imentale di Medicina e Farmacologia, Padiglione H, 4° piano, Policlinico Universitario “G. Mar- tino”, 98125 Messina, Italy. Formalin-fixed and paraffin-embedded specimens of the surgical E-mail: [email protected] samples were cut into 5-µm thick sections to perform Haema- Accepted November 18, 2008. toxylin-Eosin (H&E) stain and immunohistochemical studies.

946 JEI_08_157_Ruggeri.qxp:Layout 1 13-01-2009 9:27 Pagina 947

G-proteins in non-functioning pituitary adenomas

Table 1 - Clinical and histological features of the 22 investigated patients with non-functioning pituitary adenomas at recruitment. The table includes also the immunohistochemical (IHC) and molecular studies’ results. *Case no. 14 shows a CGT>TGT transition resulting in conversion of arginine to cysteine at codon 201. Case no. 21 shows a SNP C>G at codon 318 in the promoter region of GNA12A gene. Patient Age Sex Tumor size Cavernous sinus Bone Recurrence Histology IHC GNAS1A GNAI2 (yr) (M/F) (mm) invasiveness erosion mutation promoter (Exons 8 and 9)a SNPb 1 48 M 35×30 No Yes Yes Chromophobe NSc No No 2 57 M 25×22 Yes No Yes Chromophobe FSH +/– No No 3 61 M 20×18 No No No Acidophile FSH, LH +/– No No 4 49 M 18×16 Yes No No Chromophobe NS No No 5 63 F 15×13 No Yes No Chromophobe NS No No 6 51 M 40×30 No Yes Yes Chromophobe LH, PRL +/– No No 7 50 M 40×30 Yes Yes No Acidophile NS No No 8 64 M 25×20 Yes Yes Yes Acidophile NS No No 9 41 M 34×33 Yes Yes No Chromophobe PRL +/– No No 10 68 F 14×13 No No No Chromophobe FSH/LH +/– No No 11 68 M 35×25 Yes Yes No Chromophobe NS No No 12 46 M 38×35 No Yes No Acidophile FSH, LH, PRL+/– No No 13 63 M 24×19 No Yes No Chromophobe NS No No 14* 65 M 40×40 Yes Yes Yes Chromophobe NS CGT>TGT; R201C No 15 44 F 24×21 No No No Chromophobe NS No No 16 52 M 18×15 Yes No No Chromophobe NS No No 17 26 F 25×20 Yes Yes Yes Chromophobe NS No No 18 74 M 30×25 Yes Yes No Acidophile NS No No 19 67 F 32×28 Yes Yes No Chromophobe NS No No 20 35 F 15×14 No No No Chromophobe NS No No 21* 75 M 25×20 No No No Acidophile NS No -318 C>G 22 74 M 24×20 No No No Chromophobe PRL +/– No No

aFor seek of simplicity, data on GNAI2A mutation are not reported in table, being all patients negative. bSNP: single nucleotide polymorphism. cNS: no staining. +/–: positively stained cells less than 10%, interspersed within the negatively stained tumor. M: male; F: female.

Immunohistochemistry (IHC) for anterior pituitary hormones were separately amplified using the following primers: and α-subunit was performed, separately, by using mouse mon- GNAS1A exon 8, 5’-AGGCTGACTATGTGCCGAGCGA-3’ oclonal antibodies raised against human FSH, LH, TSH, PRL, and 5’-ACCCACGTCAAACATGCTGGTG-3’; GNAS1A ex- GH, and ACTH (all from Dako, Carpinteria, CA, USA) and on 9, 5’-ATCAGGGTCGCTGCTCAC-3’ and 5’-TG- mon©oclonal antibody against α-subunit (from Abcam, UK). CACGGGGTTCTTCTCTAT-3’; GNAI2 exon 5, 5’- Briefly,2008, tissue sections were deparaffinized Editrice in xylene, rehy- GAAATGGCA KurtisTGGGAGGGAAGG-3’ and 5’-TAAAAC- drated, and exposed to microwave heating (at 100 C for 15 CTCAGTGGGGCTGGG-3’; GNAI2A exon 6, 5’- min, in citric acid buffer, pH 6.0). Then, the sections were in- TGAAGTGGGCAAGTGTGGATG-3’ and 5’-AGGAAGAG- cubated with eachFOR antibody over PERSONALnight at 4 C. Staining was ob- GATGA USETGGACGT-3’; ONLY GNAI2A promoter, 5’-ACC- tained using the biotin-streptavidin-peroxidase method (LSAB CTCGGGTTAACAGATCC-3’ and 5’-GGGGCCAGAGGA- kit from Dako Corporation, Carpinteria, CA) and the reac- GATTAGAG-3’. tion was developed with 3,3’-diaminobenzidine (DAB, Dako). All PCR reactions were performed in a 25-µl mixture con- Negative controls included: a) omission of the primary anti- taining 20 pmol of each primer, 200 µM of each dNTP, 0.5 IU serum; b) replacement of the primary antiserum with normal of Taq Gold polymerase (Applied Biosystems) or 1.25 IU of mouse or goat serum. In each of these conditions, no staining Taq Gold for GNAI2A promoter amplification, and 30-50 ng of was evident. DNA. The conditions of the PCR were the following: a) GNAS1 exons 8 and 9: initial denaturation at 95 C for 5 min, PCR studies and DNA sequencing followed by 36 cycles of denaturation at 94 C for 30 sec, an- DNA was isolated from 5-µm formalin-fixed and paraffin-em- nealing at 55 C for 60 sec for exon 8 and 58 C for exon 9, and bedded sections, as elsewhere described (15). The sections extension at 72 C for 30 sec; after the last cycle, a final ex- were dewaxed with two xylene washes (30 min at 55 C each), tension step at 72 C for 8 min was performed; b) GNAI2 ex- one 100% ethanol wash, two 70% ethanol washes, and two ons 5 and 6: initial denaturation at 95 C for 4 min, followed distilled water washes (6 min at 55 C for each step) and then by 37 cycles of annealing at 94 C for 30 sec, extension at 58 C incubated with proteinase K (20 mg/ml) and digestion buffer for 30 sec and denaturation at 72 C for 30 sec with a final ex- [100 mM NaCl/10 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH tension step of 8 min at 72 C; c) GNAI2A promoter: initial 8.0, and 0.5% sodium dodecyl sulfate (SDS)]. denaturation at 94 C for 4 min, annealing (34 cycles) at 94 C “Hot spots” exons 8 and 9 of the GNAS1 gene, exons 5 and for 45 sec, extension at 60 C for 45 sec, and denaturation at 6 of the GNAI2 gene and the promoter region of GNAI2 72 C for 45 sec with a final extension of 8 min at 72 C. PCR

947 JEI_08_157_Ruggeri.qxp:Layout 1 13-01-2009 9:27 Pagina 948

R.M. Ruggeri, L. Santarpia, L. Curtò, et al.

amplifications were performed using GeneAmp PCR System A T G CCG T G T C 9700 (Applied Biosystems, Foster City, CA). All PCR products were run onto 2% agarose gel to establish the quality of the PCR reaction, and purified using a QIAquick PCR Purification Kit (Qiagen). The samples were direct sequenced (ABI PRISM 310 Genetic Analyzer; Applied Biosystems) using the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems), ac- cording to the manufacturer’s instructions. We used different normal pituitary DNA as negative controls.

B T G C N G T G T C RESULTS Histological, IHC, and molecular data of the 22 investigated NFPA patients are summarized in Table 1. The histological features included 16 cromophobe and 6 aci- dophile adenomas. IHC studies for anterior pituitary hormones were negative in the majority of cases (15/22 patients). Weak positive staining for FSH and/or LH and/or PRL was observed in a minority of cells, interspersed within negatively stained cells, in 7 cases (Table 1). Fig. 1 - A) Normal Sequence of GNAS1 in a case of non-functioning pituitary In the present series, we found only one case harboring the adenoma without mutation. B) Chromatogram of GNAS1 mutant sequence following GNAS1 mutation: a CGT>TGT transition, resulting in our case of non-functioning pituitary adenoma harboring the mutation arginine to cysteine at codon 201 (black arrow). in conversion of arginine to cysteine at codon 201 (Fig. 1). The patient harboring GNAS1 mutation was one of the 6 relapsing NFPA. However, he did not differ from the other patients in terms of age, tumor size, cavernous sinus invasiveness, and his- ther exons 5 and 6 of the GNAI2A gene were noted in the tological and immunohistochemical data. No other mutation same series. The patient harboring GNAS1 mutation was one in GNAS1 was found. of the 6 relapsing NFPA of the present series. Moreover, no mutations of GNAI2A gene were identified in Our data are in line with data of the literature (9, 11) and con- our 22 patients. Nevertheless, we found a silent polymorphism firm that clinically different types of pituitary tumor (like NF- (SNP) C>G at codon 318 in the promoter region of the PA and GHomas) may share common somatic mutations, GNAI2A gene. The SNP was heterozygous. According to the which probably occur in pluripotent pituitary cells, as suggest- University of California Santa Cruz (UCSC) browser ed by Tordjman and colleagues (9). Nevertheless, we confirm (http://genome.ucsc.edu) and due to the fact that the promot- that these gene mutations are not a common finding in the er represent an untranslated region (UTC) of the gene, this NFPA. Probably, mechanisms other than G proteins mutations C>G base change, obviously, does not led to amino acid se- may play a more relevant role in cAMP cascade activation, thus quence modifications. Also this patient – as the patient har- promoting pituitary tumorigenesis in this type of tumors. bouring the GNAS1A mutation – did not show any clinical, Additionally, we found a silent polymorphism (SNP) C>G at ©histological and immunohistochemical feature as compared to codon 318 in the promoter of the GNAI2A gene in one of 22 the pat2008,ients without SNP. EditriceNFPA, i.e. a fre Kurtisquency of 4.5% in our series. Although the - 318G carrier patient not displayed peculiar clinical and/or histopathological features with respect to the C carriers, we DISCUSSION FOR PERSONALthink t USEhat this finding is ONLY noteworthy. In fact, to our knowledge, GNAS1 mutations have been reported hitherto in a limited this is the first report of a variation in the GNAI2 gene in pi- number of NFPA (about 10%), which were clinically indistin- tuitary adenomas. The GNAI2A gene has been screened only guishable from GNAS1 mutations-negative NFPA. Tordjman for mutations, which have been reported in a small propor- and colleagues (9) demonstrated GNAS1A mutations in 2 of tion of NFPA (9) and in one corticotroph adenomas (12), so the 21 clinically non-functioning pituitary tumors analyzed far. Previous studies have demonstrated a low expression of Gi (10%): arginine at codon 201 altered to cysteine, and glutamine proteins in tumoral pituitary tissues and correlated it to tu- at codon 227 changed to leucine. Williamson et al. (10) iden- mor growth, but the mechanism responsible for this phe- tified the same GNAS1 mutations at codon 201 and 227 in nomenon has not been clarified (16). 2/22 (13%) of NFPA. Similarly, the two groups did not demon- Recently, the -318 C>G SNP in the GNAI2A promoter region strate GNAI2A mutations in any of the 27 NFPA studied (9), has been associated with higher systolic blood pressure and and in only 3 out of 22 NFPA, which harbor GNAI2A mutation increased risk for hypertension in the general population (17). at codon 205 (glutamine to arginine). Among these three The authors attributed this association to the decreased pro- GNAI2A-mutated tumors, two also harbored GNAS1 muta- moter activity shown by the -318G variant, as demostrated in tions (10). More recently, Kan and co-workers found the transfected HEK293 cells (17). The reduced transcriptional ac- known GNAS1 mutation at codon 201 (arginine to cysteine) tivity demonstrated in -318G carriers by Menzaghi and co- and no mutations of GNAI2A gene in 1/7 NFPA (11). workers may represent a possible cause of the decreased ex- In our 22 NFPA screened for both GNAS1 and GNAI2A mu- pression of Gi proteins reported in tumoral pituitary tissues tations, we found only one case harboring a GNAS1 mutation (16). As a result of such modifications in GNAI2 gene expres- (arginine to cysteine at codon 201), while no mutations in ei- sion, -318G carriers might have increased cAMP levels that, in

948 JEI_08_157_Ruggeri.qxp:Layout 1 13-01-2009 9:27 Pagina 949

G-proteins in non-functioning pituitary adenomas

turn, promote tumor growth. Noteworthy, the down-regula- characteristics of acromegalic patients whose pituitary tumors contain tion of GNAI2 transcription when the -318C>G SNP is ex- mutant Gs protein. J Clin Endocrinol Metab 1990, 71: 1416-20. 8. Spada A, Arosio M, Bochicchio D, et al. Clinical, biochemical, and mor- pressed in the promoter region, is probably due to specific phological correlates in patients bearing growth hormone-secreting pi- binding with the transcription factor Sp1. This is an intriguing tuitary tumors with or without constitutively activated adenylyl cyclase. issue, because Sp1 is known to enhance the transcription of J Clin Endocrinol Metab 1990, 71: 1421-6. pituitary tumor transforming gene, a regulator of cell division 9. Tordjman K, Stern N, Ouaknine G, et al. Activating mutations of the Gs that is over-expressed in NFPA (18). alpha-gene in non-functioning pituitary tumors. J Clin Endocrinol Metab In our NFPA series, we report a frequency of the -318C>G 1993, 77: 765-9. 10. Williamson EA, Daniels M, Kendall-Taylor P, Harris PE. Gs alpha and Gi SNP of 4.5% (1/22), but the not large number of patients does alpha mutations in ‘non-functioning’ pituitary tumours. Clin Endocrinol not consent of establishing a real prevalence. (Oxf) 1994, 41: 815-20. Even though we were unable of finding the polymorphic variant 11. Kan B, Esapa C, Sipahi T, et al. G protein mutations in pituitary tumors: (-318C>G GNAI2) in the National Center for Biotechnology In- a study on Turkish patients. Pituitary 2003, 6: 75-80. formation (NCBI) SNP database in order to evaluate the fre- 12. Williamson EA, Harrison D, Ince PG, Kendall-Taylor P, Harris PE. G-pro- tein mutations in human pituitary adrenocorticotrophin hormone-se- quency of this specific SNP in general population, we cannot creting adenomas. Eur J Clin Invest 1995, 25: 128-31. rule out a possible role of this polymorphic variant in the patho- 13. Boston BA, Mandel S, LaFranchi S, Bliziotes M. Activating mutation in the genesis of NFPA, because of its location. It could be better de- stimulatory guanine nucleotide-binding protein in an infant with Cushing’s fined if the real frequency and the specific characteristics will syndrome and nodular adrenal hyperplasia. J Clin Endocrinol Metab 1994, be demonstrated in an adequately large population of patients. 79: 890-3. 14. Petersenn S, Heyens M, Lüdecke DK, Beil FU, Schulte HM. Absence of so- matostatin receptor type 2 A mutations and gip oncogene in pituitary so- REFERENCES matotroph adenomas. Clin Endocrinol (Oxf) 2000, 52: 35-42. 15. Santarpia L, El-Naggar AK, Cote GJ, Myers JN, Sherman SI. Phospha tidyli - 1. Gittoes NJ. Current perspectives on the pathogenesis of clinically non- nositol 3-kinase/akt and ras/raf-mitogen-activated protein kinase pathway functioning pituitary tumours. J Endocrinol 1998, 157: 177-86. mutations in anaplastic thyroid cancer. J Clin Endocrinol Metab 2008, 93: 2. Alexander JM, Biller BM, Bikkal H, Zervas NT, Arnold A, Klibanski A. 278-84. Clinically nonfunctioning pituitary tumors are monoclonal in origin. J Clin 16. Ballaré E, Mantovani S, Bassetti M, Lania A, Spada A. Immunodetection of Invest 1990, 86: 336-40. G proteins in human pituitary adenomas: evidence for a low expression 3. Herman V, Fagin J, Gonsky R, Kovacs K, Melmed S. Clonal origin of pi- of proteins of the Gi subfamily. Eur J Endocrinol 1997, 137: 482-9. tuitary adenomas. J Clin Endocrinol Metab 1990, 71: 1427-33. 17. Menzaghi C, Paroni G, De Bonis C, et al. The -318 C>G single-nucleotide 4. Asa SL, Ezzat S. The pathogenesis of pituitary tumours. Nat Rev Cancer polymorphism in GNAI2 gene promoter region impairs transcriptional 2002, 2: 836-49. activity through specific binding of Sp1 transcription factor and is associ- 5. Levy A, Lightman S. Molecular defects in the pathogenesis of pituitary tu- ated with high blood pressure in Caucasians from Italy. J Am Soc Nephrol mours. Front Neuroendocrinol 2003, 24: 94-127. 2006, 17 (Suppl 2): S115-9. 6. Lania A, Mantovani G, Spada A. Genetics of pituitary tumours: focus on 18. Zhang X, Horwitz GA, Heaney AP, et al. Pituitary tumor transforming G-protein mutations. Exp Biol Med (Maywood) 2003, 228: 1004-17. gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab 7. Landis CA, Harsh G, Lyons J, Davis RL, McCormick F, Bourne HR. Clinical 1999, 84: 761-7.

© 2008, Editrice Kurtis FOR PERSONAL USE ONLY

949