The Genetics of Pituitary Adenomas

The genetic landscape of pituitary adenomas (PAs) is diverse and many of the identified cases remain of unclear pathogenetic mechanism. Germline genetic defects account for a small percentage of all patients and may present in the context of relevant family history. Defects in AIP (mutated in Familial Isolated Pituitary Adenoma syndrome or FIPA), MEN1 (coding for menin, mutated in Multiple Endocrine Neoplasia type 1 or MEN 1), PRKAR1A (mutated in Carney complex), GPR101 (involved in X-Linked Acrogigantism or X-LAG), and SDHx (mutated in the so called “3 P association” of PAs with pheochromocytomas and paragangliomas or 3PAs) account for the most common familial syndromes associated with PAs. Tumor genetic defects in USP8, GNAS, USP48 and BRAF are some of the commonly encountered tissue-specific changes and may explain a larger percentage of the developed tumors. Somatic (at the tumor level) genomic changes, copy number variations (CNVs), epigenetic modifications, and differential expression of miRNAs, add to the variable genetic background of PAs.


Introduction
Pituitary adenomas (PAs) are common lesions in the adult population presenting in 15-20% of cadavers or radiologic findings and constitute approximately 10% of all intracranial tumors [1,2]. PAs are rarer in the pediatric population identified in 0.2% of children undergoing brain imaging [3]. Although most of the identified PAs are incidental findings without the need for intervention, some may present as clinically significant because they secrete hormones or cause symptoms from compression or invasion of surrounding tissues [4].
The etiology of PAs is diverse, and more than half of them do not have an identified genetic cause. In certain cases, however, germline or somatic genetic defects are associated with the formation of PAs. Additional genetic changes found in PAs, such as copy number variations (CNVs), methylation changes and miRNA abnormalities have also been investigated as being potentially involved in the pathogenesis, presentation and behavior of these tumors, especially with regard to aggressiveness and response to treatment.
This review focuses on the current knowledge of the genetic findings in PAs at the germline (Tables 1 and 2) and somatic (Table 3) level. Additional information on the genomic profile of PAs can derive from expression studies, methylation analyses and miRNA changes, which are briefly mentioned in the current review. GH excess with onset of symptoms in most cases younger than 2 years of age.

Neurofibromatosis type 1 NF1
Autosomal dominant syndrome presenting with neurofibromas, skin findings (café-au-lait macules and freckling), Lisch (iris) nodules, optic pathway gliomas with consequent precocious puberty and GH excess, and other rare tumors. PAs can be classified based on their functional status ( Figure 1). In adults, most PAs are prolactin (PRL)-secreting, while non-functioning adenomas represent the second most common type. In children, depending on the study population, ACTH-and PRL-secreting adenomas are the most commonly encountered subtypes [5]. PAs may also be classified as sporadic (95% of cases), when relevant family history is not present, or familial (5% of cases), when additional family members with similar or relevant disorders are identified [6].
PAs can be classified based on their functional status ( Figure 1). In adults, most PAs are prolactin (PRL)-secreting, while non-functioning adenomas represent the second most common type. In children, depending on the study population, ACTH-and PRL-secreting adenomas are the most commonly encountered subtypes [5]. PAs may also be classified as sporadic (95% of cases), when relevant family history is not present, or familial (5% of cases), when additional family members with similar or relevant disorders are identified [6].

Familial Isolated Pituitary Adenomas (FIPA)
The term Familial Isolated Pituitary Adenoma (FIPA) is used to describe families with at least two members with pituitary adenomas, with or without other abnormalities [7]. FIPA accounts for 2-4% of all patients with PAs [7]. Tumors within a family can be homologous (of the same subtype) or heterologous (of different subtypes) [7].
Aryl hydrocarbon receptor interacting protein (AIP) gene mutations were described as a cause of FIPA in 2006, and they are currently thought to account for almost 15% of all FIPA cases; this number is even higher (up to 75%) when families with GH-secreting adenomas are selected [8,9]. AIP gene defects are less common in sporadic pituitary adenomas identified in up to 8% of all cases [10,11]. However, when younger patients (<30 years old) with macroadenomas, or children and adolescents <18 years old are selected, up to 20.5% of cases with sporadic PAs may harbor an AIP mutation [12][13][14][15]. The exact mechanism of the pituitary tumorigenesis with AIP mutations is not clear. Some evidence suggests that there is interaction of AIP in the synthesis of cAMP, and decreased AIP activity leads to aberrant cAMP levels, which then affect several pathways involving cell proliferation [16].
Patients with AIP mutations commonly present with GH-secreting adenomas; the second most common presentation is that of GH/PRL co-secreting adenomas. It has been often described that PAs with AIP mutations have a more aggressive presentation compared to AIP-negative tumors: patients often present in younger age, with larger tumors, higher chance of invasion of surrounding tissues, higher risk for apoplexy, and less chance for control with one intervention, especially in the pediatric population [17][18][19][20][21][22].

Familial Isolated Pituitary Adenomas (FIPA)
The term Familial Isolated Pituitary Adenoma (FIPA) is used to describe families with at least two members with pituitary adenomas, with or without other abnormalities [7]. FIPA accounts for 2-4% of all patients with PAs [7]. Tumors within a family can be homologous (of the same subtype) or heterologous (of different subtypes) [7].
Aryl hydrocarbon receptor interacting protein (AIP) gene mutations were described as a cause of FIPA in 2006, and they are currently thought to account for almost 15% of all FIPA cases; this number is even higher (up to 75%) when families with GH-secreting adenomas are selected [8,9]. AIP gene defects are less common in sporadic pituitary adenomas identified in up to 8% of all cases [10,11]. However, when younger patients (<30 years old) with macroadenomas, or children and adolescents <18 years old are selected, up to 20.5% of cases with sporadic PAs may harbor an AIP mutation [12][13][14][15]. The exact mechanism of the pituitary tumorigenesis with AIP mutations is not clear. Some evidence suggests that there is interaction of AIP in the synthesis of cAMP, and decreased AIP activity leads to aberrant cAMP levels, which then affect several pathways involving cell proliferation [16].
Patients with AIP mutations commonly present with GH-secreting adenomas; the second most common presentation is that of GH/PRL co-secreting adenomas. It has been often described that PAs with AIP mutations have a more aggressive presentation compared to AIP-negative tumors: patients often present in younger age, with larger tumors, higher chance of invasion of surrounding tissues, higher risk for apoplexy, and less chance for control with one intervention, especially in the pediatric population [17][18][19][20][21][22].

Multiple Endocrine Neoplasia Syndromes
Multiple endocrine neoplasia (MEN) syndromes are autosomal dominant disorders that present with combination of tumors in at least two endocrine systems [23].
Interestingly, PAs in MEN1 have higher chance of co-secreting multiple hormones compared to MEN1-negative patients, in up to 39% of cases [27]. PAs in MEN1 are considered more aggressive and at higher risk for resistance to treatment, especially in children with large prolactinomas [32]. Somatic MEN1 gene mutations are not commonly found in sporadic pituitary tumors [33,34].
MEN4 (OMIM#610755) is caused by CDKN1B gene mutations, which codes for a cyclin-dependent kinase (p27) that regulates cell cycle and progression from G1 to S phase of mitosis [43]. MEN4 is a rare genetic syndrome, accounting for approximately 1.5-3% of patients clinically classified as MEN1, without genetic defects in MEN1 gene [44,45]. Of the reported index cases to date, five patients have been diagnosed with PAs (two with GH-secreting, two with ACTH-secreting, and one with non-functioning adenoma) [44][45][46][47][48][49]. CDKN1B gene mutations in FIPA have been a rare entity and are described in approximately 2% of AIP-negative FIPA kindreds [50]. To date, only one patient with isolated sporadic GH-secreting PA has been described in the literature [51].

Carney Complex
Carney complex (CNC) describes the constellation of myxomas, spotty skin pigmentation, and endocrine overactivity [52,53]. Germline mutations of the PRKAR1A gene are responsible for more than 70% of cases of CNC (OMIM#160980), whereas few patients may harbor defects at another locus on chromosome 2p16 [54,55]. PRKAR1A codes for the type 1 alpha regulatory subunit of the protein kinase A (PKA) tetramer. Inactivating mutations of PRKAR1A lead to dissociation of the regulatory from the catalytic subunit, resulting in aberrant activity of PKA and phosphorylation of downstream targets, leading to cell proliferation and tumor formation [53].
Amongst the common endocrine abnormalities described in CNC are pituitary hyperplasia and PAs that are often associated with GH and/or PRL excess [56]. Although definite adenomas are present in 15% of patients, abnormal GH response to various stimuli, such as glucose or TRH, is present in almost 75% of patients [57,58]. Pituitary involvement in CNC is thought to be a progressive disorder with normal pituitary tissue progressing to somato(mammo)troph hyperplasia and subsequently, to distinct tumor formation [58]. Thus, multiple adenomas may be present synchronous or metachronous in the same patient [59]. Once diagnosed, GH excess is usually slowly progressive and although transsphenoidal resection of PAs may be attempted, there is high risk for recurrence. Medical therapy is often offered to the patients and sometimes, partial or complete hypophysectomy may be needed for control of disease [58,59].
Although CNC was thought to be associated almost exclusively with GH and /or PRL excess, most recently, two patients with Cushing disease (CD) have also been described, showing that other pituitary cell lineages may be rarely affected [60,61]. Somatic PRKAR1A gene changes are not reported in sporadic PAs [62,63].

McCune-Albright Syndrome (MAS)
The classic presentation of MAS (OMIM#174800) includes the triad of polyostotic fibrous dysplasia (FD), café-au-lait pigmentation and precocious puberty [64]. The disease is the result of postzygotic activating mutations of the GNAS1 gene product, the cAMP-regulating protein Gsalpha (Gs α ) [65]. It is now well recognized that additional endocrine overactivity disorders, other than precocious puberty, are part of the condition, including GH excess. GH excess in MAS presents in almost 21% of patients and may contribute to worsening of FD, vision and hearing loss, and macrocephaly [66]. For that reason, prompt medical therapy is recommended in all patients, while surgical intervention is not frequently attempted due to thick bones, the potential for worsening of skull FD, as well as the need for partial or total hypophysectomy [64].
In a large study of 143 pediatric patients with gigantism, 10% of patients had microduplication of Xq26.3, two in familial cases and ten in sporadic adenomas [70]. Compared to patients without an identified genetic etiology, patients presented younger than the remaining patients, were more likely to co-secrete PRL, and had less risk of invasion of surrounding tissues [70]. In a cohort of 18 patients with X-LAG, mean age at diagnosis was 41 months and most patients had a macroadenoma identified on MRI, although no giant adenoma (by criteria of diameter more than 4 cm) was reported [67]. X-LAG is generally recognized as an aggressive disease given the difficulty to control GH excess. Most of the patients require multiple interventions (surgical and medical), and sometimes, subtotal or total hypophysectomy may be necessary. Radiation therapy is not generally helpful [67,71].

3 P Association (3PAs)
The three P association or 3PAs is another recently identified condition involving the combination of pituitary adenomas, pheochromocytomas (PHEO) and/or paragangliomas (PGL) [72]. Xekouki et al. described that up to 75% of familial cases with 3PAs harbor pathogenic variants in the SDHx (SDHB and SDHD) genes [72]. Succinate dehydrogenase is part of the mitochondrial complex II and plays a significant role in energy production through the Krebs cycle and the respiratory chain through electron transfer [73]. Additional studies have further contributed in the genetic spectrum of the disease, by identifying additional genetic causes of the association (SDHA, SDHAF2, VHL, MEN1, RET and MAX) [74][75][76][77][78]. The presenting pituitary tumors are more commonly PRL-or GH-secreting or non-functioning adenomas, but they are thought to be more aggressive in presentation and more likely to be resistant to standard therapy [77].
A rare but almost pathognomonic manifestation of DICER1 syndrome is pituitary blastoma (PitB). PitBs differ from PAs in terms of their histological characteristics (since these are embryonic tumors) but given the potential for hormone production, they should be considered in the context of pituitary tumors. ACTH-secreting PitBs, although extremely rare, are almost always associated with DICER1 mutations [81,82]. They are always diagnosed in infants and toddlers, less than 24 months of age [82]. Their management involves, in most cases, surgical removal, while at least one of the patients was treated with TMZ and radiation therapy. Four of the eleven reported patients died from complications of their disease, while at least two patients experienced recurrence [82]. Since PitBs are a rare association of DICER1 syndrome, routine screening with imaging or biochemical studies is not currently recommended for patients with DICER1 syndrome [83].
Recently, a case of isolated PRL-secreting adenoma in a 50-year old patient has been reported. Although histologic evaluation of the identified sellar tumor was not available, and the patient did not respond to medical therapy, this report expands the potential implications of DICER1 in adult-onset pituitary tumorigenesis [84].

Less Common Germline Genetic Defects Potentially Associated with PAs
NF1 (OMIM#613675) is an autosomal dominant syndrome caused by mutations or deletions in the NF1 gene, reported in 1:3500 births [85]. NF1 codes for neurofibromin which acts as a Ras-GTPase-activating protein; neurofibromin deficiency leads to constitutive activation of Ras-dependent pathways, namely Ras/Raf/MEK and Ras/PI3K/TSC/mTOR [85]. GH excess presents in 5.5-10.9% of patients with NF1 and optic pathway gliomas (OPGs), and it is thought to result from suppression of the hypothalamic somatostatin tone, while GHRH overactivity from OPGs has also been postulated in certain cases [90,91]. As a result, there is diffuse pituitary hyperplasia and no distinct adenoma is usually identified. Only one patient with a GH-secreting adenoma has been reported to date [92]. However, NF1 should remain in the differential diagnosis when evaluating a patient with acromegaly/gigantism.
Recently, Hernandez-Ramirez et al. described four potentially pathogenic variants in the CABLES1 gene in patients with CD. CABLES1 interacts with cyclin-dependent kinase 3 and genetic defects lead to impaired inhibition of cell growth [93]. CNVs and methylation defects of other cyclin-dependent kinases (CDK2A and CDKN2C, coding for p16 and p18) have been reported in studies of PAs [94,95]. Additionally, a somatic mutation in DKC1 gene, coding for dyskeratin, associated with X-Linked dyskeratosis congenita when present in germline, has been described in a patient with a non-functioning pituitary adenoma. The mutation is potentially associated with decreased p27 levels or defects in rRNA modification [96].
Finally, few patients with diagnosis of PA and additional genetic syndromes have been reported in the literature, for example, a patient with Beckwith-Wiedemann syndrome (loss of methylation of imprinting critical region 2 on chromosome 11p) and ACTH-secreting adenoma, two family members with Cantu syndrome (ABCC9) and non-functioning adenomas, a patient with autosomal dominant polycystic kidney disease (PKD1) and acromegaly, and a patient with X-linked adrenal hypoplasia (due to a DAX1 defect) and ACTH-secreting adenoma [97][98][99][100]. The etiologic association of the pathogenesis of PA with the identified germline genetic defect is not confirmed in most of them, however, potential relationship cannot be excluded.

Somatic Changes
As mentioned above, somatic changes of genes involved in identified syndromes when present in germline may be identified exclusively in the tumor level in certain cases. For example, GNAS mutations are present in up to 50% of GH-secreting adenomas. Below we present genetic defects reported mainly in the tumor level of PAs.
The prognosis of patients with somatic USP8 mutations differs in various studies. Some reports of increased risk for recurrence of patients with somatic USP8 mutations have not been replicated by others [104,106,107]. However, the potentially aggressive behavior of this genetic defect is evident in the patient with germline USP8 defect, described recently by Cohen et al. [108]. The patient had severe recurrent CD difficult to control with surgical and medical therapies [108].

USP48 and BRAF
With the understanding of the importance of USP8 in corticotropinomas, further studies for other genes related to the MAPK pathway have been published. Recently, USP48 and BRAF have been reported as additional target genes, with somatic variants of BRAF gene in 16% of patients and of USP48 gene in 23% of USP8-negative corticotropinomas [109]. Of interest, variants in these genes were not detected in other pituitary tumors, suggesting a specific defect in the POMC regulation [109].

GNAS
Somatic GNAS mutations are common in sporadic GH-secreting adenomas presenting in up to 50% of these tumors [110,111]. Mutations are usually located at codons 201 and 227, and lead to activation of the G sa subunit of GNAS, leading to increased adenylate cyclase activity, similarly to the defect seen in patients with MAS. Because of paternal imprinting of GNAS in the pituitary gland, mutations need to occur at the maternal allele to result in pituitary tumorigenesis [112]. A limited number of ACTH-secreting adenomas with somatic GNAS gene defects has been reported, showing that there may be a rare association with CD [113,114].

The Phosphoinositide 3-Kinase (PI3K)/AKT Pathway
The PI3K/AKT pathway regulates several cellular functions, including cell survival, growth, proliferation and metabolism [115]. Gene defects, including pathogenic variants and amplifications, in PIK3CA have been isolated in several cancers [116]. Studies in PAs identified a potential role in pituitary tumorigenesis as well with a frequency of somatic defects in 2.3-12.1% of all tumors, with higher incidence in invasive PAs [117,118].

The p53 Tumor Suppressor Gene
TP53 codes for a tumor suppressor protein that has important implication in human carcinogenesis. Although p53 expression in pituitary tumors is associated with more aggressive behavior, several studies have failed to show a significant role of genetic defects of TP53 in pituitary tumorigenesis [119][120][121]. Few cases of atypical PAs and pituitary carcinomas with somatic TP53 defects have only been reported to date [122][123][124].

Copy Number Variations (CNVS) at the Chromosomal Level
Several studies have investigated the presence of CNVs in PAs. Although no recurrent CNV has been clearly noted, CNVs in chromosomes 1p and 11p have been reported in several occasions [125,126].
Recently, Bi et al. described a different profile of genomic aberrations: in a cohort of 42 PAs, a group of "disrupted" tumors, defined as large CNVs encompassing on average 39% of the tumor genome, were noted. These tumors were more likely to be functional adenomas, without evidence of more aggressive behavior [127,128]. Similar results were further described by other groups in GH-secreting tumors [129,130]. Our group also supported these studies by identifying a subgroup of patients with CD that harbored large genomic aberrations, involving up to 59% of the tumor genome. These tumors were associated with larger size and higher risk for cavernous sinus invasion [131].

Epigenetic Modifications of Pituitary Adenomas
The understanding of pathogenesis, differentiation and presentation of PAs has been further advanced through studies of epigenetic modifications.
Genome-wide methylation analyses of PAs have identified changes in methylation profile between normal pituitary tissue and PAs and attempted to clarify the differences between various subtypes of PAs [132,133]. Although the results have not been always consistent, changes in the methylation pattern of tumor suppressor and cell-cycle regulating genes (such as CASP8, tp73, RASSF1, Rb, p16, p18, SFN and other) or genes involved in the behavior of the tumors and their potential to invade tissues, like cell adhesion genes have been described [95,[133][134][135][136]. Methylation changes also correlate with certain tumor characteristics, such as their size, functional status, and invasion of surrounding tissues, but again, results have not been replicated in all studies [94,133,135,[137][138][139].
Growth factors have also been studied in relation to pituitary development and tumorigenesis [140]. FGFR2, and specifically, the predominat isoform FGFR2-IIIb) is downregulated in half of the pituitary adenomas, possibly due to increased promoter methylation, with potential implication for the expression of certain cyclin dependent kinases (such as p21 and p27) [141]. Furthermore, FGFR2 interacts with melanoma-associated antigen-3 (MAGE-A3) complex which further regulates p53 expression and leads to disordered cell cycle regulation [142]. FGFR4, on the other hand, is overexpressed in PAs, with potential implication in tumorigenesis [143].
Histone modifications and chromatin changes may also explain some of the PA characteristics, leading to altered expression of important cell-cycle genes (like Rb, p21 and p27) [142]. Examples of histone modifications and chromatin remodeling include the case of Ikaros, where expression of a certain isoform results in histone 3 acetylation and activation of the Bcl-XL promoter, leading to increased survival of pituitary cells [144,145].

miRNAs
miRNAs are small non-coding RNAs that are involved in cell cycle regulation and cancer pathogenesis via control of expression of various genes [146,147]. Over the last 10 years, studies on differentially expressed miRNAs in PAs compared to normal pituitary tissues have identified miRNAs with potential interest. For example, downregulation of miR-326, miR-432, miR-570, and miR-603, has been described in GH-secreting adenomas. These miRNAs potentially target the HMGA2 and E2F1 genes and their downregulation leads to increased expression and results in increased cell proliferation [148]. Several additional miRNAs, either upregulated or downregulated targeting genes affecting cell proliferation, apoptosis, invasion and even response to therapy, have been described, with potential implications for diagnostic, prognostic and therapeutic purposes [149][150][151].

Conclusions
The genomic landscape of PAs remains complex. Germline and somatic genetic defects contribute to our understanding of their pathogenesis, but do not explain the majority of them. Additional information derived from other gene regulation mechanisms should be considered in combination to describe the full picture of pituitary tumorigenesis.
Author Contributions: C.T. and C.A.S. contributed equally to this manuscript. All authors have read and agree to the published version of the manuscript.

Conflicts of Interest:
Stratakis holds patents on technologies involving PRKAR1A and related genes, and his laboratory has received research funding support by Pfizer Inc, unrelated to this project.