Landscape of somatic mutations in sporadic GH-secreting pituitary adenomas

in European Journal of Endocrinology
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  • 1 Department of Internal Medicine I, Endocrinology and Diabetology Unit, Central Laboratory, Institute of Human Genetics, Comprehensive Cancer Center Mainfranken, Medizinische Klinik and Poliklinik IV, Core Unit Systems Medicine, Endocrinology Unit, Institute of Pharmacology and Toxicology and Bioimaging Center, Department of Neurosurgery, Neurosurgery, Institute of Human Genetics, Endocrine and Diabetes Unit, University Hospital, University of Wuerzburg, Oberduerrbacherstrasse 6, 97080 Wuerzburg, Germany

Context

Alterations in the cAMP signaling pathway are common in hormonally active endocrine tumors. Somatic mutations at GNAS are causative in 30–40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear.

Aim

Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations.

Design

Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome sequencing (n=36).

Results

By targeted sequencing, known activating mutations in GNAS were detected in five cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole-exome sequencing identified 132 protein-altering somatic mutations in 31/36 tumors with a median of three mutations per sample (range: 1–13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, seven genes involved in cAMP signaling pathway were affected in 14 of 36 samples and eight samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No significant correlation between genetic alterations and the clinical data was observed.

Conclusion

This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway, calcium signaling might be involved in the pathogenesis of these tumors.

Abstract

Context

Alterations in the cAMP signaling pathway are common in hormonally active endocrine tumors. Somatic mutations at GNAS are causative in 30–40% of GH-secreting adenomas. Recently, mutations affecting the USP8 and PRKACA gene have been reported in ACTH-secreting pituitary adenomas and cortisol-secreting adrenocortical adenomas respectively. However, the pathogenesis of many GH-secreting adenomas remains unclear.

Aim

Comprehensive genetic characterization of sporadic GH-secreting adenomas and identification of new driver mutations.

Design

Screening for somatic mutations was performed in 67 GH-secreting adenomas by targeted sequencing for GNAS, PRKACA, and USP8 mutations (n=31) and next-generation exome sequencing (n=36).

Results

By targeted sequencing, known activating mutations in GNAS were detected in five cases (16.1%), while no somatic mutations were observed in both PRKACA and USP8. Whole-exome sequencing identified 132 protein-altering somatic mutations in 31/36 tumors with a median of three mutations per sample (range: 1–13). The only recurrent mutations have been observed in GNAS (31.4% of cases). However, seven genes involved in cAMP signaling pathway were affected in 14 of 36 samples and eight samples harbored variants in genes involved in the calcium signaling or metabolism. At the enrichment analysis, several altered genes resulted to be associated with developmental processes. No significant correlation between genetic alterations and the clinical data was observed.

Conclusion

This study provides a comprehensive analysis of somatic mutations in a large series of GH-secreting adenomas. No novel recurrent genetic alterations have been observed, but the data suggest that beside cAMP pathway, calcium signaling might be involved in the pathogenesis of these tumors.

Introduction

Pituitary tumors represent ∼15% of all primary intracranial lesions. Growth hormone (GH)-secreting pituitary adenomas are the second most frequent type of hormone-producing pituitary tumors, after prolactin-secreting adenomas (1). Excessive secretion of GH causes gigantism during childhood and acromegaly in adults, with significant morbidity due to clinical complications involving cardiovascular, respiratory, and metabolic systems (2, 3).

The monoclonal origin of most pituitary adenomas indicates that these tumors derive from the replication of a single cell that acquired growth advantage. The latter has been suggested to result from genetic or epigenetic alterations, leading to activation of proto-oncogenes or inactivation of tumor suppressor genes (4, 5). However, despite intensive investigations, little is known about the genetic causes of pituitary adenomas. The only mutations identified to date in a significant proportion (30–40%) of sporadic GH-secreting adenomas occur in the gene encoding the α subunit of the stimulatory G-protein (GNAS) (6, 7, 8, 9). These somatic activating mutations (gsp mutations), found in codon 201 and 227, prevent hydrolysis of GTP, leading to a constitutive activation of the cAMP pathway, which in somatotrophs and in other endocrine cells acts as a mitogenic signal (10, 11). In somatotrophs, the GNAS transcript is expressed mainly from the maternal allele, due to tissue-specific paternal imprinting (12, 13). Consistently, gsp mutations in sporadic GH-secreting adenomas are found on the maternal allele (14), and partial loss of this imprinting is present in tumors negative for gsp mutations (15), further supporting the involvement of GNAS locus in pituitary tumorigenesis. So far, the screening for mutations in other G-protein subunits in pituitary tumors has given negative results (16, 17, 18).

Genetic alterations in other genes involved in cAMP signaling have been identified as the cause of other endocrine tumors. A reduced expression and/or function of the protein kinase A (PKA) regulatory subunit type Iα (PRKAR1A) due to loss-of-function mutations, leading to an abnormal cAMP pathway activation, causes GH-secreting pituitary adenomas in Carney complex, an autosomal dominant familial syndrome (19, 20). To date, mutations of PRKAR1A gene have been rarely found in sporadic pituitary tumors (21, 22), although a reduced PRKAR1A expression resulting from increased proteasomal degradation has been described in sporadic GH-secreting tumors (10). Reduced cAMP degradation caused by mutations in PDE11A and PDE8B, coding for members of the phosphodiesterase (PDE) family, have been involved in adrenocortical hyperplasia, adenomas, and cancer as well as in testicular germ cell tumors (23, 24). However, genetic variants of PDE11A4 contribute only marginally to the development of GH-secreting adenomas (25). Recently, mutations affecting the gene encoding the catalytic subunit α of the PKA (PRKACA) have been reported in a large proportion of cortisol-secreting adrenocortical adenomas (26, 27, 28, 29, 30), resulting in an increased PKA activity (31). Nevertheless, no hot spot mutations of PRKACA have been identified in a large cohort of GH-secreting adenomas (32). Finally, a recurrent somatic mutation in the GPR101 gene, which encodes an orphan G-protein-coupled receptor (GPCR), has been recently reported in some adults with acromegaly (4% of cases) (33).

In addition, epidermal growth factor receptor (EGFR) overexpression has been described in hormonally active pituitary adenomas (34) and role for epidermal growth factor (EGF) and its receptor in the development and/or progression of pituitary tumors has been hypothesized (35). Dominant mutations in the deubiquitinase USP8 gene that promote activation of EGFR signaling have been also found in adrenocorticotropin (ACTH)-secreting pituitary adenomas by exome sequencing (36). Finally, germline mutations of genes such as the aryl hydrocarbon receptor-interacting protein (AIP), the menin (MEN1), and the p27 (CDKN1B) have been reported in genetic syndromes associated with acromegaly (i.e., familial-isolated pituitary adenoma and multiple endocrine neoplasia type 1 and 4) and in a low percentage of young acromegalic patients (37).

Recently, Valimäki et al. investigated a small group of 12 patients with GH-secreting adenomas by whole-genome sequencing and single nucleotide polymorphism (SNP) array and did not find any novel recurrent genetic alteration (38). Aim of the present study was to perform a comprehensive genetic characterization of a large series of GH-secreting adenomas to identify novel genetic alterations potentially involved in tumorigenesis and/or in clinical outcome. To this aim, we used both targeted direct sequencing of GNAS, PRKACA, and USP8 genes and next-generation exome sequencing.

Subjects and methods

Tissue samples, patients, and clinical annotations

Sporadic GH-secreting adenomas without familial or syndromic presentation were recruited in the present study. Accordingly, a total of 81 fresh frozen tumors were collected from four different participating European centers. If available, corresponding peripheral blood was also collected for the analysis. Inclusion criteria for participating in the study were a certified histological diagnosis of benign GH-secreting adenomas and available clinical data. The DNA was isolated as described previously (39). Qualitative and quantitative evaluation of the DNA was assessed by electrophoresis in a 1% agarose gel and spectrophotometrically at 260 nm respectively. At the first screening, 14 tumor samples have been excluded due to insufficient DNA quality so that the final series included a total of 67 GH-secreting adenomas. Among them, the tumor samples were subdivided into two groups according to the availability of corresponding leukocyte DNA essential for next-generation whole-exome sequencing. Thus, 31 tumor samples underwent targeted direct sequencing for the analysis of selected genes (GNAS, PRKACA, and USP8) (Group 1), while the remaining 36 cases with corresponding leukocyte DNA were investigated by whole-exome sequencing (Group 2).

Clinical parameters, such as sex, age at diagnosis, date of surgery, tumor size, GH and IGF1 levels, presence of acromegaly-related complications, and follow-up data, were collected for all patients at the local centers. All the patients gave written informed consent, and the study was approved by the ethics committee at each participating institution.

Targeted and whole-exome sequencing and data analysis

For the targeted sequencing analysis, we focused on gene domains harboring alterations known or supposed biologically relevant in endocrine active tumors, i.e. known gain-of-function GNAS mutations (codon 201 and 227), mutations in the catalytic domain of the PRKACA (exon 7 and 8), and in the 14-3-3 binding domain and the microtubule-interacting and transport-domain (exons 1, 2, and 3) of the USP8, which is reported to be involved in regulating USP8 catalytic function. The primers used for the targeted direct sequencing were generated with the Program Primer3Plus (39).

The complete list of the primers is reported in the Supplementary Table 1, see section on supplementary data given at the end of this article. In brief, PCR was performed on 1 μl of diluted DNA (2 ng/μl) in a final volume of 25 μl containing 1.5 mM MgCl2, 0.2 μM of each primer, 200 μM dNTPs and 1 U Taq DNA polymerase. The reaction was started with an initial 95  °C denaturation step for 3 min, followed by 30 cycles of denaturation at 93  °C (20 s), annealing at 58  °C (30 s) and elongation at 72  °C (1 min). Direct sequencing of PCR products was performed using the QuickStart cycle sequencing kit (ABSciex Four Valley drive Concord, Ontario, Canada) on a CEQ8000 DNA analyzer (ABSciex).

For the next-generation sequencing, exomes were enriched in solution and indexed with the use of the SureSelect XT human all exon 50 Mb kit, version 5 (Agilent Technologies, Santa Clara, CA, USA). Sequencing was performed as paired-end reads of 100 bp on a HiSeq2500 systems (Illumina, San Diego, CA, USA) generating 8–14 Gb of sequence and an average depth of coverage between 110× and 170× on target regions. More than 95% of the target regions were covered 20 times or more. Pools of 12 indexed libraries were sequenced on four lanes. Image analysis and base calling were performed with the use of real-time analysis software (Illumina). Reads were aligned against the human assembly hg19 (GRCh37) using the Burrows-Wheeler Aligner tool (BWA v 0.7.5a). Variant detection was done as described earlier (26).

Somatic variants have been evaluated by Polymorphism Phenotyping v2 tool (PolyPhen-2) (40) and scale invariant feature transform (SIFT) algorithm (http://sift.jcvi.org/index.html) (41). An unsupervised complete linkage clustering including the most relevant somatic mutations was performed by the Hamming distance as a similarity metric. The Gene Set Enrichment Analysis (GSEA) software was used for the gene enrichment and the functional annotation (Broad Institute, MSigDB database v5.0; http://software.broadinstitute.org/gsea) (42). A canonical pathway analysis (1330 gene set) and a gene family analysis were also performed with the same software.

Statistical analysis

Median, interquartile range, and frequency were used as descriptive statistics. IGF1 values were expressed as percentage of the upper limit of the normal range (%ULN). The Fisher's exact test or the χ2-test were used to investigate dichotomic variables, while a two-sided t-test (or non-parametric test) was used to test continuous variables. A non-parametric Kruskal–Wallis test, followed by the Bonferroni post hoc test, was used for multiple comparisons among several groups for non-normal distributed variables. Correlations and 95% CIs between the total number of mutations and different clinical parameters were evaluated by linear regression analysis. Statistical analyses were performed using the GraphPad Prism (version 5.0, La Jolla, CA, USA) and SPSS (PASW version 21.0, SPSS, Inc.) software. P values <0.05 were considered as statistically significant.

Results

Targeted DNA sequencing (Group 1)

A total of 31 patients affected by GH-secreting adenomas were included in this group. Minimum clinical data were available for 17 of them (10 males and 7 females; median age: 46 years, range: 19–64; 16 macroadenomas and 1 microadenoma; median basal GH levels: 24.3 ng/ml, range: 2.3–333; median IGF1 %ULN: 391, range: 266–590).

We observed the presence of known activating GNAS mutations in five out of 31 evaluated samples (16.1% of cases), i.e. a p.Arg201Cys substitution in four samples and a p.Gln227Leu in one sample. We did not identify any mutation in all the evaluated exons of PRKACA and USP8 (Table 1). However, we detected different polymorphisms in the USP8 gene: exon 1 (rs3131575 T/G heterozygous in eight cases and homozygous in one case, rs11632697 G/C heterozygous in 14 cases and homozygous in one case, and rs11632708 C/T heterozygous in 13 cases and homozygous in one case) and 14-3-3 binding domain (rs11638390 A/G heterozygous p.T739A in 14 cases and homozygous in one case) (Table 1). Allele frequencies did not differ significantly from frequencies reported in dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) (Supplementary Table 2, see section on supplementary data given at the end of this article).

Table 1

List of genetic alterations at targeted sequencing of 31 GH-secreting pituitary adenomas. PRKACA exons 7 and 8 and USP8 exons 2 and 3 showed no sequence variants and are not included.

Tumor ID (Group 1)GNASUSP8Ex 1USP814-3-3 domain
1–16WTrs3131575 heterozygousWT
1–18WTrs11632697 heterozygousrs11638390 heterozygous
1–20WTWTWT
1–24WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–25WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–26WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–28WTWTWT
1–29WTWTWT
1–31WTWTWT
1–32WTrs11632697 homozygous, rs11632708 homozygousrs11638390 homozygous
1–33WTrs11632697 heterozygous, rs11632708 heterozygous, rs3131575 heterozygousrs11638390 heterozygous
1–35WTWTWT
1–37WTrs11632697 heterozygous, rs11632708 heterozygous, rs3131575 heterozygousrs11638390 heterozygous
1–38WTWTWT
1–39WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–40p.Q227L heterozygousWTWT
1–43WTWTWT
1–44WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–46WTrs3131575 heterozygousWT
1–48WTWTWT
1–50p.R201C heterozygousrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–51WTrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–52WTWTrs11638390 heterozygous
1–53p.R201C heterozygousrs11632697 heterozygous, rs11632708 heterozygousrs11638390 heterozygous
1–54p.R201C heterozygousWTWT
1–55rs121913494 heterozygousrs11632697 heterozygous, rs11632708 heterozygous, rs3131575 heterozygousrs11638390 heterozygous
1–57WTrs3131575 heterozygousWT
1–58p.R201C heterozygousrs11632697 heterozygous, rs11632708 heterozygous, rs3131575 heterozygousrs11638390 heterozygous
1–60WTrs3131575 homozygousWT
1–61WTrs3131575 heterozygousWT
1–62WTWTWT

Bold values represent protein altering mutations.

Next-generation exome sequencing (Group 2)

The histopathological and clinical parameters for the patients included in this analysis are reported in the Table 2. At the whole-exome sequencing, we identified a total of 132 protein-altering somatic mutations in 36 samples, resulting in a median of three somatic mutations in exonic regions per sample (range: 0–13). The genetic alterations included 109 missense and seven nonsense mutations, 12 frameshift, two direct splicing, and two indel variations. According to the PolyPhen-2, 39 mutations were classified as probably damaging, 25 as possibly damaging, and 41 as benign. The entire list of the somatic mutations including localization, gene symbols, and transcripts is reported in the Supplementary Table 3, see section on supplementary data given at the end of this article.

Table 2

Clinical and genetic data of the 36 patients affected by GH-secreting pituitary adenomas evaluated by next-generation exome-sequencing (Group 2).

AllNo mutationsLow number of mutation (≤3)High number of mutations (>3)P
Clinical data at the time of diagnosisa
 n3651417
 Sex (M/F)16/181/46/68/9NS
 Age (years)47.8±19.032.4±28.044.2±7.054.1±17.5NS
 Tumor size (mm)18.8±9.,613.0±3.520.6±10.019.2±11.2NS
  Micro/MACRO2/321/41/111/16NS
 GH levels (μg/l)
  Basal24.3±27.,950.4±58.529.7±28.218.2±14.4NS
  Post-OGTT nadir21.6±20.,815.2±16.029.7±31.920.3±18.9NS
 IGF1 levels (%UNL)334±152312±165282±122372±158NS
 Pituitary deficiencies (yes/no)5/290/54/81/160.07
 Co-secretion with prolactin (yes/no)7/271/41/115/12NS
 Diabetes (yes/no)10/221/43/96/11NS
 Hypertension (yes/no)10/221/43/96/11NS
 Cardiac disease (yes/no)3/290/51/112/15NS
Clinical data after surgery
 Additional radiotherapy (yes/no)3/270/50/123/12NS
 Additional drug therapy (yes/no)9/193/24/82/9NS
 Biochemical remission (yes/no)b9/71/35/23/4NS
Genetic data
 Total number of mutations17501.6±0.96.3±3.1<0.001
 GNAS mutations (n – %)11 (29.7%)05 (35.7%)6 (33%)NS

M, male; F, female; micro, microadenoma; MACRO, macroadenoma; OGTT, oral glucose tolerance test; and %ULN, percentage of the upper normal limit.

The clinical data from two patients are not available.

Biochemical remission evaluated 3–6 months after surgery.

We identified a subgroup of patients without any mutation (negative, n=5), a subgroup with a low number of mutations (n 1–3; n=14), and a subgroup with a high number of mutations (n>3; n=17) (Fig. 1). No significant correlation was observed between the total number of mutations and the evaluated clinical data, such as sex, age, tumor size and extension, and the initial GH levels.

Figure 1
Figure 1

Total number of somatic mutations in the 36 GH-secreting pituitary tumors evaluated by next-generation exome sequencing (Group 2). The tumors affected by mutations in GNAS are represented with red bars. The numeration of the GH-secreting adenomas is consecutive and do not correspond to the tumor identification number.

Citation: European Journal of Endocrinology 174, 3; 10.1530/EJE-15-1064

The most frequent genetic alterations were the known gain-of-function mutations in the GNAS gene. Specifically, they were detected in 11 cases (31.4% of total, ten of them being females), encoding p.Arg201Cys substitution in seven samples, p.Arg201His in two samples, and p.Gln227Leu in two samples. No difference was observed in total number of mutations between the tumors with or without GNAS mutations (Fig. 1). No further genetic alterations were found in more than one sample in this series. Even comparing the list of the mutated genes with that of a recent paper on whole-genome sequencing in 12 GH-secreting adenomas (37), no additional recurrent somatic genetic alterations were observed.

However, some non-recurrent heterozygous somatic variants were observed in genes encoding GPCR, such as the chemokine receptor 10 (CCR10) and the olfactory receptor OR51B4, which are coupled to the Gs protein (activation of the cAMP signaling pathway), and the M3 muscarinic cholinergic receptor (CHRM3), which functions through Gq (activation of the inositol trisphosphate/calcium signaling pathway). Moreover, other non-recurrent alterations were found in genes coding for proteins involved in cAMP signaling pathway other than GNAS, such as the α2 catalytic subunit of the AMP-activated protein kinase (PRKAA2), the G-protein-coupled receptor kinase 3 (GRK3, alias ADRBK2), and the A1 subunit of the lysosomal H+ ATPase (ATP6V0A1). Taken together, the mutations in genes involved in the cAMP signaling affected 14/36 samples (38.9% of total). Among them, nine samples presented only GNAS mutations, two samples mutations at GNAS and other genes of the cAMP signaling and three only mutations in other genes encoding GPCR or other members of the cAMP signaling. The corresponding details are reported in the Table 3.

Table 3

List of somatic mutations in genes associated with the cAMP signaling pathway or in the calcium signaling/metabolism detected in 36 GH-secreting pituitary adenomas (Group 2; the RefSeq annotation does not provide any annotated transcript for the position chr6:119532096, corresponding ENSEMBL identification ‘ENST00000368466’).

Gene symbolGene nameBase changeProtein changeNumber of samples
cAMP signaling pathway
 GNASGNAS complex locusc.601C>Tp.Arg201Cys7
c.602G>Ap.Arg201His2
c.584A>Tp. Gln227Leu2
 PRKAA2Protein kinase, AMP-activated, α2 catalytic subunitc.1132C>Gp.Pro378Ala1
 ADRBK2Adrenergic, β, and receptor kinase 2c.1976G>Ap.Arg659His1
 ATP6V0A1ATPase, H+ transporting, and lysosomal V0 subunit A1c.94C>Ap.Leu32Ile1
 CCR10Chemokine (C–C motif) receptor 10c.691C>Tp.Gly231Ser1
 CHRM3Cholinergic receptor and muscarinic 3c.1244A>Gp.Asp415Gly1
 OR51B4Olfactory receptor, family 51, subfamily B, and member 4c.619C->Tp.Asp207Asn1
Calcium signaling
 CACNA1HCalcium channel, voltage-dependent, T type, and α1H subunitc.1175C>Tp.Ser392Leu1
 CAPN1Calpain 1 and large subunitc.380A>Gp.Asn127Ser1
 DMDDystrophinc.960G>Cp.Ser320Arg1
 GRIN2BGlutamate receptor, ionotropic and N-methyl d-aspartate 2Bc.1894C>Ap.Val632Leu1
 JPH2Junctophilin 2c.1111C>Ap.Glu371*1
 MAN1A1Mannosidase, α, class 1A, and member 1c.AG>>AANA1
 PCDH11XProtocadherin 11 X-linkedc.3725C>Gp.Ala1242Gly1
 PROCA1Protein interacting with cyclin A1c.749C>Gp.Arg250Pro1
 SLIT2Slit homolog 2c.4190G>Ap.Cys139Tyr1
 SPTA1Spectrin, α, erythrocytic 1c.7201G>Ap.Arg2401*1
 TESCTescalcin10_16delAGTGGGC, frameshift, 1175370711

NA, not applicable.

Corresponds to non-sense mutations according to the HVGS recommendations.

Finally, a number of altered genes associated at different levels with the Ca2+ signaling and metabolism (i.e., involving both extra and intracellular compartment) were observed in eight cases (22.2% of total). They consisted in the α1H subunit of the voltage-dependent T type calcium channel (CACNA1H), the large subunit of the calpain 1 (CAPN1), the dystrophin (DMD), the NMDA ionotropic glutamate receptor 2B (GRIN2B), the junctophilin 2 (JPH2), the mannosidase α class 1A (MAN1A1), the X-linked protocadherin 11 (PCDH11X), the protein interacting with cyclin A1 (PROCA1), the slit homolog 2 (SLIT2), the erythroid α1 spectrin (SPTA1), and the tescalcin (TESC) (Table 3).

An unsupervised clustering including all the somatic mutations in genes involved in the cAMP pathway or in the Ca2+ signaling was performed. The results including the relationship with the total number of somatic mutations and clinical data are shown in the Fig. 2.

Figure 2
Figure 2

Overview of the somatic mutations at genes involved in the cAMP signaling (i.e., CCR10, OR51B4, CHRM3, GNAS, PRKAA2, GRK3, and ATP6V0A1) or in the calcium signaling (i.e., CACNA1H, CAPN1, DMD, GRIN2B, JPH2, MAN1A1, PCDH11X, PROCA1, SLIT2, SPTA1, TESC) in GH-secreting adenomas evaluated by next-generation exome sequencing (n=36) and relationship with the total number of somatic mutations, sex, basal GH levels, and tumor extension at the time of diagnosis. Age: child <18 years, young ≤50 years (median), old >50 years; tumor size: macro=macroadenoma and micro=microadenoma; tumor extension: extra=extrasellar and intra=intrasellar; and GH: low ≤15 μg/l (median) and high >15 μg/l.

Citation: European Journal of Endocrinology 174, 3; 10.1530/EJE-15-1064

Concerning the correlation with the clinical data, the patients with mutations in genes of the cAMP signaling pathway were mostly females (10/14, 71%), while those with mutations in genes associated with the Ca2+ signaling were mostly males (5/7, 71%) and those with other kinds of mutations were equally distributed between the two sexes (50%, P<0.001 by Kruskal–Wallis test for multiple comparisons) (Figs 2 and 3). A trend to a lower total number of mutations and younger age was observed in the group of patients without alterations of the cAMP or Ca2+ signaling in comparison with the other two groups (Figs 2 and 3). No significant differences in terms of tumor size and basal GH or IGF1 levels have been found.

Figure 3
Figure 3

Relationship between the genetic alterations observed at the exome sequencing (i.e. mutations in genes member of the cAMP pathway, of the calcium signaling or in others) and clinical data (i.e. total number of somatic mutations in upper panel, age in the middle panel, and sex in the lower panel) in 36 evaluated GH-secreting adenomas.

Citation: European Journal of Endocrinology 174, 3; 10.1530/EJE-15-1064

Functional annotation and pathway analysis

The gene enrichment analysis in the entire series identified a total of 117 altered genes associated with a gene ontology term. Several altered genes resulted to be associated with developmental biological processes (Supplementary Table 4, see section on supplementary data given at the end of this article). The canonical pathway analysis recognized no significant overlaps. The gene family analysis showed the presence of one cytokine/growth factor (SLIT2), seven protein kinases (ADRBK2, CDK10, CHUK, EPHA8, PRKAA2, SCYL1, and TESK1), four known oncogenes (GNAS, KDM5A, SH3GL1, and STIL) and two tumor suppressor genes (SETD2 and TSC2) among the mutated genes.

Discussion

The present study offers a comprehensive genetic characterization of a large cohort of 67 GH-secreting pituitary adenomas. We aimed to identify novel molecular markers potentially involved in tumorigenesis and/or in clinical outcome. To this end, we first performed targeted sequencing of GNAS, PRKACA, and USP8 genes in order to evaluate the presence of mutations in these genes in GH-secreting adenomas, finding only known GNAS gene mutations. By whole-exome sequencing, only a limited number of genetic alterations have been detected in the 36 evaluated samples. This finding is consistent with the low mitotic activity of pituitary tumors and with previous small studies on both non-functioning (n=7) (43) and GH-secreting pituitary adenomas (n=12) (37). Moreover, no recurrent somatic mutations have been observed, except the known alterations at the GNAS gene, similarly to a previous report on a small series of GH-secreting adenomas (37). In particular, no somatic mutations have been also detected at the gene GPR101, probably due to the low reported frequency of this mutations (11/248 cases) (33), and, at both the exome sequencing and the targeted sequencing, we did not find any mutations of the PRKACA and USP8 genes. These findings further confirm that both these genetic alterations are not involved in the pathogenesis of GH-secreting adenomas (32, 36).

Interestingly, several non-recurrent alterations affected other genes involved in the cAMP signaling besides GNAS (see Table 2). These findings further support the view that deregulation of cAMP pathway is the most important pathogenetic mechanism in GH-secreting adenomas. Furthermore, a number of genes associated with the Ca2+ signaling (see Table 2) were altered. These findings are in agreement with another recent study on whole-genome alterations in 12 GH-secreting adenomas (37). This is consistent with the notion that binding of growth hormone-releasing hormone to its receptor activates not only the stimulatory subunit α of the G-protein (Gα-S, cAMP-dependent pathways) but also Gα-I, Gβ, and Gγ, leading to release of intracellular free Ca2+, which then further triggers secretion of GH (44, 45). Moreover, ATP, which is co-released with pituitary hormones, induces an increase in free Ca2+ in pituitary cells (46). These data strongly suggest that dysregulation of the calcium signaling might be an important co-signal in somatotrophs and potentially involved in pituitary tumorigenesis. However, its biological role needs to be better investigated in future functional studies.

It has been suggested that tumors might be very heterogeneous with few mutations in common. Instead, different genes acting through the same molecular pathways may contribute to tumor formation (47). Therefore, it is possible that at least some of these low-frequency GH-secreting tumor variants present tumor-promoting mutations. Alternatively, they may present other types of molecular alterations not detectable by exome sequencing (i.e., mutations in non-coding intronic chromosomal regions).

In conclusion, we found no novel recurrently mutated genes in a large series of GH-secreting pituitary adenomas. However, our and previous genetic findings suggest that beside cAMP pathway, also different pathways, such as Ca2+ signaling, may play an important role in the pathogenesis of these tumors.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/EJE-15-1064.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The study was supported by grants IZKF Wuerzburg (B-281 to D Calebiro and M Fassnacht), by the ERA-NET ‘E-Rare’ (01GM1407B to M Fassnacht) and by the Associazione Italiana Ricerca Cancro, Milan (AIRC) (IG-15 507 to G Mantovani).

Author contribution statement

C L Ronchi, B Allolio, M Reincke and M Fassnacht conceived the idea of the study. C L Ronchi developed the protocol of the study, coordinated the collection of the tissue materials and the clinical data, performed the statistical analysis, and wrote the first draft of the paper; E Peverelli, G Mantovani, and A Spada, provided the tumor tissue and the corresponding blood samples and contributed to wrote the paper; S Herterich performed the targeted sequencing analysis; I Weigand, D Calebiro, and S Sbiera contributed to the protocol of the study and to the data analysis; S Appenzeller performed the clustering and the heatmap; J Honegger, M Reincke, M Buchfelder, and J Flitsch provided the tumor tissue and the corresponding blood samples; T M Strom performed the whole-exome sequencing analysis including the filtering and the first data analysis; and M Fassnacht contributed to the coordination of the study and to write the paper. All the authors reviewed and approved the final version of the manuscript.

Acknowledgements

The authors are grateful to Mrs Michaela Bekteshi for expert technical support.

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  • View in gallery

    Total number of somatic mutations in the 36 GH-secreting pituitary tumors evaluated by next-generation exome sequencing (Group 2). The tumors affected by mutations in GNAS are represented with red bars. The numeration of the GH-secreting adenomas is consecutive and do not correspond to the tumor identification number.

  • View in gallery

    Overview of the somatic mutations at genes involved in the cAMP signaling (i.e., CCR10, OR51B4, CHRM3, GNAS, PRKAA2, GRK3, and ATP6V0A1) or in the calcium signaling (i.e., CACNA1H, CAPN1, DMD, GRIN2B, JPH2, MAN1A1, PCDH11X, PROCA1, SLIT2, SPTA1, TESC) in GH-secreting adenomas evaluated by next-generation exome sequencing (n=36) and relationship with the total number of somatic mutations, sex, basal GH levels, and tumor extension at the time of diagnosis. Age: child <18 years, young ≤50 years (median), old >50 years; tumor size: macro=macroadenoma and micro=microadenoma; tumor extension: extra=extrasellar and intra=intrasellar; and GH: low ≤15 μg/l (median) and high >15 μg/l.

  • View in gallery

    Relationship between the genetic alterations observed at the exome sequencing (i.e. mutations in genes member of the cAMP pathway, of the calcium signaling or in others) and clinical data (i.e. total number of somatic mutations in upper panel, age in the middle panel, and sex in the lower panel) in 36 evaluated GH-secreting adenomas.