Steroidogenic Factor 1 Overexpression and Gene Amplification Are More Frequent in Adrenocortical Tumors from Children than from Adults
Madson Q. Almeida, Ibere Cauduro Soares, Tamaya C. Ribeiro, Maria Candida B. V. Fragoso, Lidiane V. Marins, Alda Wakamatsu, Rodrigo A. Ressio, Mirian Y. Nishi, Alexander A. L. Jorge, Antonio M. Lerario, Venancio A. F. Alves, Berenice B. Mendonca, and Ana Claudia Latronico
Unidade de Endocrinologia do Desenvolvimento (M.Q.A., T.C.R., M.C.B.V.F., M.Y.N., A.A.L.J., A.M.L., B.B.M., A.C.L.), Laboratório de Hormônios e Genética Molecular/(Laboratório de Investigação Médica 42) da Disciplina de Endocrinologia do Hospital das Clínicas, and Divisão de Anatomia Patologia, Laboratório de Patologia Hepática/LIM14 do Hospital das Clínicas (I.C.S., L.V.M., A.W., R.A.R., V.A.F.A.), Faculdade de Medicina da Universidade de São Paulo, 05403-900 São Paulo, Brasil
Background: Steroidogenic factor 1 (SF-1) is a key determinant of endocrine development and function of adrenal cortex. SF-1 overexpression and gene amplification were previously demon- strated in a small group of pediatric adrenocortical tumors.
Objective: Our objective was to determine the frequency of SF-1 protein expression and gene amplification in a large cohort of pediatric and adult adrenocortical tumors.
Patients: SF-1 protein expression was assessed in a cohort of 103 adrenocortical tumors from 36 children and 67 adults, whereas gene amplification was studied in 38 adrenocortical tumors (17 from children).
Methods: Tissue microarray, multiplex ligation-dependent probe amplification, and quantitative real-time PCR were used.
Results: A strong nuclear SF-1 expression was detected by tissue microarray in 56% (20 of 36) and 19% (13 of 67) of the pediatric and adult adrenocortical tumors, respectively (P= 0.0004). Increased SF-1 copy number was identified in 47% (eight of 17) and 10% (two of 21) of the pediatric and adult adreno- cortical tumors, respectively (P = 0.02). All adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five (29%) pediatric adrenocortical tumors without SF-1 amplification. The frequency of SF-1 overexpression and gene amplification was similar in adrenocortical adenomas and carcinomas.
Conclusion: We demonstrated a higher frequency of SF-1 overexpression and gene amplification in pediatric than in adult adrenocortical tumors, suggesting an important role of SF-1 in pediatric adrenocortical tumorigenesis. (J Clin Endocrinol Metab 95: 1458-1462, 2010)
A drenocortical carcinoma is a rare malignancy with incompletely understood pathogenesis and poor prognosis (1). However, a remarkably high annual inci- dence of adrenocortical tumors has been reported in chil-
dren younger than 15 yr from Southern Brazil, where a high prevalence of a germline mutation of the P53 tumor suppressor (p.R337H) was reported (2, 3). Besides im- portant differences in prognosis, adrenocortical tumor-
doi: 10.1210/jc.2009-2040 Received September 22, 2009. Accepted December 11, 2009. First Published Online January 15, 2010
Abbreviations: CT, Cycle threshold; MLPA, multiplex ligation-dependent probe amplifica- tion; qRT-PCR, quantitative real-time PCR; SF-1, steroidogenic factor 1; TMA, tissue microarray.
igenesis also has distinct patterns between children and adults (4, 5).
Steroidogenic factor 1 (SF-1) is an orphan member of the nuclear receptor family of transcription factors and plays an important role in endocrine function, including the regulation of steroid hydroxylases, development and function of the adrenal cortex, and male sexual differen- tiation (6). SF-1 maps to 9q33.3, a chromosomal region associated with amplification in pediatric adrenocortical tumors (7). Sf-1 dosage regulates compensatory adrenal growth after unilateral adrenalectomy in mice (8). Fur- thermore, increased SF-1 dosage promotes cell prolifera- tion and triggers tumorigenesis in mice (9).
An increased SF-1 copy number was detected in eight of nine adrenocortical tumors diagnosed in Brazilian chil- dren using fluorescence in situ hybridization, suggesting an association between SF-1 gene amplification and pe- diatric adrenocortical tumorigenesis (10). All these adre- nocortical tumors also had SF-1 protein overexpression (11). The aim of our study was to investigate SF-1 expres- sion and gene amplification in a larger cohort of pediatric and adult adrenocortical tumors and evaluate the prog- nostic value of these findings.
Patients and Methods
The study was approved by the Ethics Committee of Hospital das Clinicas, Sao Paulo, Brazil, and informed written consent was obtained from all patients and/or parents. SF-1 protein expres- sion was assessed in adrenocortical tumors obtained from 36 children (30 adenomas and six carcinomas) and 67 adults (40 adenomas and 27 carcinomas) (Table 1). Thirty-eight samples from these sporadic adrenocortical tumors (17 children and 21 adults; 22 adenomas and 16 carcinomas) were used to analyze SF-1 amplification and gene expression. Endocrine syndromes were diagnosed in all children with adrenocortical tumors (iso- lated Cushing syndrome 8.3%, virilization 58.3%, and mixed 33.3%). Cushing syndrome was present in 48% of adults with
adrenocortical tumors, whereas 25% of adrenocortical tumors in this group were nonfunctioning. Virilization or mixed syn- drome occurred in 9 and 18% of adrenocortical tumors in adults, respectively. The diagnosis of malignancy in the pediatric group was established by an advanced tumor stage (III or IV) and/or poor clinical outcome, whereas Weiss criteria were used in adult adrenocortical tumors.
Tissue microarray (TMA) and immunohistochemical analysis
Representative areas of the 109 adrenocortical tumors were identified on hematoxylin- and eosin-stained slides and marked on paraffin donor blocks. The spotted areas of donor blocks were punched (1.0 mm punch) and mounted into a recipient paraffin block using a precision microarray instrument (Beeccher Instru- ments, Sun Prairie, WI). One set of three slides was selected (one slide from each TMA block of the triplicate) for staining with SF-1 monoclonal antibody (N1665 clone; PPMX Perseus Pro- teomics, Komaba, Japan) (12, 13). An immunoperoxidase im- munohistochemical modified method with humid heat antigen retrieval was used as previously described (14). Loss of tissue sample occurred in 5.5% of the spots. Therefore, SF-1 immu- noreactivity was analyzed in 103 tumor tissue samples. Two investigators (ICS and LVM), who were unaware of clinical data, independently evaluated SF-1 staining. The staining intensity and the percentage of positive tumor nuclei were calculated for each specimen to obtain a final semiquantitative H score, as previously described (15). The median H score value of 13 nor- mal adrenals was chosen as the cutoff point for separating tu- mors with strong and weak SF-1 staining. The intra-observer variability was 8.4 ± 2.5%. The inter-observer agreement was 0.89. The mean of the two evaluations was taken for statistical analysis.
Multiplex ligation-dependent probe amplification (MLPA)
SF-1 copy number was determined using the SALSA MLPA kit P185-B1 Intersex (MRC-Holland, Amsterdam, The Nether- lands), which contains five probes for SF-1 gene (exons 1 and 3-6). Genomic DNA from adrenocortical tumors and five nor- mal adrenals was isolated using standard procedures. MLPA was performed as previously described (16). The tumor sample nor-
| TABLE 1. Clinical characteristics of 103 patients with adrenocortical tumors according to SF-1 staining | ||||
|---|---|---|---|---|
| Children (n = 36) | Adults (n = 67) | |||
| Weak/negative SF-1 staining (n = 16) | Strong SF-1 staining (n = 20) | Weak/negative SF-1 staining (n = 54) | Strong SF-1 staining (n = 13) | |
| Age (yr) | 3.4 ± 1.2 | 3.8 ± 0.8 | 36.6 ± 1.7 | 39.1 ± 4.8 |
| Sex (female to male) | 3:1 | 2.3:1 | 6.8:1 | 12:1 |
| Follow-up (months) | 116.7 ± 21.4 | 72.5 ± 11.2 | 67.8 ± 8.4 | 53.5 ± 18.5 |
| Clinical presentation | ||||
| Cushing | 2 (12%) | 1 (5%) | 25 (46%) | 7 (54%) |
| Virilizing | 7 (44%) | 14 (70%) | 6 (11%) | 0 |
| Mixed | 7 (44%) | 5 (5%) | 10 (19%) | 2 (15%) |
| Nonfunctioning | 0 | 0 | 13 (24%) | 4 (31%) |
| Diagnosis | ||||
| Adenoma | 13 (81%) | 17 (85%) | 33 (61%) | 7 (54%) |
| Carcinoma | 3 (19%) | 3 (15%) | 21 (39%) | 6 (46%) |
malized peak area was then divided by the average normalized peak area from normal adrenals. Dosage quotient areas outside the range 0.70-1.3 were considered abnormal.
Quantitative real-time PCR (qRT-PCR)
SF-1 amplification was also analyzed by qRT-PCR using SYBR Green in an ABI 7700 Sequence Detection System (Ap- plied Biosystems, Foster City, CA). The PCR cycling conditions were as follows: 2 min at 95 C, 40 cycles of 95 C for 15 sec and 60 C for 30 sec, and a final step at 72 C for 30 sec. The primer sequences for SF-1 and NPC-1 genes are available in Supplemental Table 1 (published as supplemental data on The Endocrine So- ciety’s Journals Online web site at http://jcem.endojournals.org). A cycle threshold (CT) value in the linear range of amplification was selected for each sample in triplicate and normalized to NPC-1. SF-1 dosage was determined using the 2-AACT method. The normalized value (ACT) for each tumor sample was then compared with the mean ACT of five normal adrenals to produce a fold change ratio (normal = 1) and multiplied by 2 to generate a copy number (normal = 2).
After surgical resection, tumor fragments were immediately frozen in liquid nitrogen and stored at -80 C until total RNA extraction using the Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was generated using the High Capacity kit (Applied Biosys- tems). Quantitative real-time PCR was performed using TaqMan Gene Expression Assays according to the manufacturer’s instruc- tions. The assay IDs were SF-1, Hs00610436_m1, and B-actin, 43263. The relative expression levels were analyzed using the 2-44CT method. The mean expression of the target genes in a pool of 61 normal adrenals (Clontech, Palo Alto, CA) was assigned an expression value of 1.0, and fold change in the SF-1 expression levels was determined for each tumor sample.
Statistical analysis
All statistical analyses were performed with the SPSS 16.0 (SPSS, Chicago, IL). Weighted K-coefficient of agreement was used to evaluate the agreement between the observers. Categor- ical variables were compared using x2 test. Continuous data are expressed as mean ± SEM. Differences in expression levels were analyzed by the two-tailed Mann-Whitney U test. P values <0.05 were considered significant.
Results
The frequency of adrenocortical tumors showing strong SF-1 staining was 56% (20 of 36) and 19% (13 of 67) in children and adults, respectively (x2 = 12.1; P = 0.0004) (Table 1 and Fig. 1). Pediatric adrenocortical tumors with strong SF-1 staining were more associated with isolated virilizing syndrome than pediatric adre- nocortical tumors with weak SF-1 staining (70 vs. 44%, respectively) (Table 1).
On the other side, Cushing syndrome (isolated or as- sociated with virilization) was more frequent in pediatric adrenocortical tumors with weak SF-1 staining when compared with SF-1-positive tumors in children (56 vs. 10%, respectively). In adults, the frequency of nonfunc-
A
B
C
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tioning tumors was not significantly different between ad- renocortical tumors with weak and strong SF-1 staining (24 vs. 31%, respectively). In addition, SF-1 immunore- activity was similar in adrenocortical adenomas and car- cinomas diagnosed in children and adults (Table 1).
SF-1 gene amplification was detected in eight (47%) of 17 pediatric adrenocortical tumors and in two (10%) of 21 adult adrenocortical tumors (x2 = 5.02; P = 0.02) (Supplemental Fig. 1). SF-1 amplification was detected in both adrenocortical adenomas (n = 5) and carcinomas (n = 5) (supplemental Table 2). Among the 10 cases with increased SF-1 copy number, nine tumors showed three copies and one had four copies of the SF-1 gene. An in- creased copy number of SF-1 was detected by both MLPA and qRT-PCR in five of 10 adrenocortical tumors, whereas in the remaining adrenocortical tumors, SF-1 am- plification was demonstrated only by qRT-PCR (n = 4) or MLPA (n = 1). The SF-1 mRNA levels were significantly higher in adrenocortical tumors associated with increased SF-1 gene copies when compared with adrenocortical tu- mors without gene amplification (fold change, 2.3 ± 0.52 vs. 1.16 ± 0.18, respectively; P = 0.001). In addition, all adrenocortical tumors with SF-1 gene amplification showed a strong SF-1 staining, whereas most of the tumors (61%) without SF-1 amplification displayed a weak or negative staining (P = 0.0008). Interestingly, a strong SF-1 staining was identified in five pediatric adrenocor- tical tumors (four adenomas and one carcinoma) with- out SF-1 amplification. The R337H P53 mutation was
previously identified in seven of 10 patients (five chil- dren and two adults) with amplification of SF-1 in ad- renocortical tumors (3).
SF-1 deletion was detected only in a cortisol-secreting adrenocortical adenoma diagnosed in a 37-yr-old patient. Decreased mRNA levels and a negative immunoreactivity for SF-1 were demonstrated in this adrenocortical tumor with SF-1 deletion.
Discussion
SF-1 is a key determinant of endocrine development, and its expression is mainly restricted to the gonads, adrenal cortex, anterior pituitary, and hypothalamus (6). The in- creased SF-1 dosage modulates the expression of tran- scripts involved in cell cycle and apoptosis (9). High fre- quency of SF-1 amplification and overexpression was previously demonstrated in a small group of pediatric ad- renocortical tumors associated with 9q gain (10, 11). Here, we investigated protein expression and gene ampli- fication of SF-1 in a larger cohort of pediatric tumors as well as in a cohort of adult adrenocortical tumors. The frequency of SF-1 nuclear overexpression was signifi- cantly higher in adrenocortical tumors diagnosed in chil- dren than in adults. Additionally, an increased copy number of SF-1 was identified in 47% of pediatric adre- nocortical tumors, whereas this finding was detected in only 10% of adult adrenocortical tumors. Therefore, our data confirm the importance of SF-1 dosage in an ex- panded group of pediatric adrenocortical tumors.
Adrenocortical tumors with SF-1 amplification showed high SF-1 mRNA levels and protein expression. Interest- ingly, SF-1 nuclear overexpression was identified in pedi- atric adrenocortical tumors without SF-1 amplification. Indeed, posttranslational modifications are known to in- fluence the activity and transcriptional capacity of SF-1 (17, 18). Phosphorylation-dependent SF-1 activation is likely mediated by the MAPK signaling pathway (17). Re- cently, it was demonstrated that SF-1-mediated transcrip- tion in adrenocortical cancer cells is mediated through cyclin-dependent kinase 7 (CDK7)-induced phosphoryla- tion (18). Cdk7 overexpression was identified in a wide range of cancer cell lines (19). Therefore, additional mo- lecular mechanisms might modulate SF-1 expression and/or activity in childhood adrenocortical tumors with- out SF-1 amplification.
SF-1 expression was previously demonstrated to be high in virilizing adrenocortical tumors and low in aldo- steronomas (20). In this study, a strong SF-1 staining was associated with a higher frequency of isolated virilizing syndrome in children with adrenocortical tumors, whereas Cushing syndrome was more frequent in pediatric adre-
nocortical tumors with weak SF-1 expression. In adults, the hormonal status of adrenocortical tumors with strong, and weak SF-1 staining was similar. Additionally, SF-1 expression was not different between functioning and nonfunctioning adrenocortical tumors.
Increased SF-1 dosage promotes proliferation of hu- man adrenocortical cells as well as controls the expression of transcripts involved in the cell cycle, apoptosis, and cell adhesion (9). Although SF-1 gene has been associated with pediatric adrenocortical tumorigenesis, its prognostic value was not previously investigated (10, 11). In this study, SF-1 nuclear immunoreactivity was similar in ad- renocortical adenomas and carcinomas from children and adults, suggesting that this factor plays a role in both be- nign and malignant tumor pathogenesis.
In conclusion, overexpression and increased copy num- ber of SF-1 were identified mainly in adrenocortical tu- mors diagnosed in children than in adults. These findings indicate a more important role of SF-1 in pediatric adre- nocortical tumorigenesis. In addition, SF-1 expression was similar in adrenocortical adenomas and carcinomas diagnosed in both pediatric and adult patients.
Acknowledgments
We thank Dr. Maria Claudia Nogueira Zerbini from the De- partment of Pathology, School of Medicine, University of Sao Paulo, for her collaboration to obtain normal adrenal tissue samples.
Address all correspondence and requests for reprints to: Madson Q. Almeida, M.D., Unidade de Endocrinologia do Des- envolvimento e Laboratório de Hormônios e Genética Molecu- lar (Laboratório de Investigação Médica 42), Hospital das Clíni- cas da Faculdade de Medicina da Universidade de São Paulo, Av. Dr. Enéas de Carvalho Aguiar, 155, 20 andar Bloco 6, 05403- 900 São Paulo, SP, Brasil. E-mail: madsonalmeida@usp.br.
This work was supported in part by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Grants 05/04726-0 and 06/00244-3 (to M.Q.A.) and by Conselho Nacional de Des- envolvimento Científico e Tecnológico (CNPq) Grants 300209/ 2008-09 (to A.C.L.), 307951/06-5 (to A.A.L.J.), and 301339/ 2008-09(to B.B.M.).
Disclosure Summary: We declare no duality of financial in- terest or direct or indirect conflict of interest on the part of any author of this manuscript.
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