Pathogenesis, clinical features, and treatment of plurihormonal pituitary adenoma

Plurihormonal pituitary adenoma (PPA) is a type of pituitary tumor capable of producing two or more hormones and usually presents as an aggressive, large adenoma. As yet, its pathogenesis remains unclear. This is the first study to systematically summarize the underlying pathogenesis of PPA. The pathogenesis is related to plurihormonal primordial stem cells, co-transcription factors, hormone co-expression, differential gene expression, and cell transdifferentiation. We conducted a literature review of PPA and analyzed its clinical characteristics. We found that the average age of patients with PPA was approximately 40 years, and most showed only one clinical symptom. The most common manifestation was acromegaly. Currently, PPA is treated with surgical resection. However, recent studies suggest that immunotherapy may be a potentially effective treatment.

Plurihormonal pituitary adenomas were previously considered rare.Recent application of immunohistochemistry to pituitary tumors has shown that many pituitary adenomas, potential source of pituitary adenomas (De Ameida et al., 2010).Radian et al. found that GH-and thyroid-stimulating hormone (TSH)secreting cells share common progenitor cell expression.This suggests that TSH adenomas originate from pituitary stem cells that differentiate into GH, PRL, or TSH cells, which may play a role in the pathogenesis of TSH-secreting PPA (Teramoto et al., 2004;Vankelecom and Roose, 2017).Matsuno et al. studied the expression of pituitary hormones including glycoprotein hormone mRNA in GH and PRL adenomas using non-isotope in situ hybridization.They found that some cells in GH adenomas expressed multiple hormone genes, mainly ACTH, FSH, and LH.These results suggest that some GH adenomas may originate from adult pituitary stem cells, which are precursors of various hormone-expressing cells (Matsuno et al., 1995(Matsuno et al., , 1996)).For example, Kovacs et al. reported a case of PPA-producing GH and ACTH.Through immunohistochemical analysis, fluoroscopic electron microscopy, immunoelectron microscopy, and in situ hybridization, the tumor was found to be composed of two independent cell types.Moreover, one cell population synthesized GH, and the other synthesized ACTH.The authors proposed that PPA may originate from undifferentiated stem cells that can differentiate into many different cell types and that this multidirectional differentiation could explain the pathogenesis of PPA (Kovacs et al., 1998).
In this study, we summarized the underlying pathogenesis (Figure 1).A growing number of studies have identified the presence of PSC in the pituitary gland.PSC can grow into adenomas under conditions such as activation of oncogenes and inactivation of oncogenes (Carreno et al., 2017;Laporte et al., 2020;Würth et al., 2020;Winningham and Camper, 2023).Under the regulation of different transcription factors, they differentiate into different cells and secrete different hormones, which ultimately lead to PPA.In addition, we found that the WNT, TGFβ, NOTCH, HIPPO and SHH pathways may be associated with adult pituitary stem cell tumorigenesis (Gaston-Massuet et al., 2011;Lodge et al., 2019;Würth et al., 2020;Winningham and Camper, 2023).(Figure 2) Further basic research on adult pituitary stem cells is needed to explore their role in the development, progression, and recurrence of PPA.

Co-transcription factor
Previous studies identified several transcription factors that regulate the transformation of cellular precursors into mature secretory cells, including T-pit, Pitx1, Pitx2, Prop-1, Pit-1, SF-1, NeuroD1, and GATA-2 (Parvin et al., 2017;Sjöstedt et al., 2017;Ellsworth and Stallings, 2018;Mete et al., 2018;Cheung and Camper, 2020;Hickman et al., 2021).Pitx1 is a universal transcription factor found in all types of pituitary cells and all types of pituitary adenomas.Pitx1 acts synergistically with NeuroD1/2, SF-1, and Pit-1 to activate POMC/ACTH, FSH-LH, and GH-PRL-TSH transcription, respectively (Szeto et al., 1999;Lamolet et al., 2001).Matsuno et al. described a GH-PRL-ACTH pituitary tumor and demonstrated the use of immunohistochemical staining, in situ hybridization, and cell culture techniques to generate GH, PRL, ACTH, and Pit-1 expression.They believed that in addition to Pit-1, there may be unknown transcription factors that simultaneously express GH, PRL, and ACTH (Matsuno et al., 1996).Shigeyuki et al. found that GH and ACTH are expressed in the same cell in one GH-ACTH adenoma, suggesting that GH is produced in ACTH-derived adenoma cells and that Pit-1 and NeuroD1 are expressed in the nucleus of the same adenoma cells.This suggests that the production of multiple cell lineage hormones in the same cell may be due to abnormal expression patterns associated with the transcription factors in pituitary tumors.The synergistic mechanism of NeuroD1 and Pit-1 is of great scientific significance and requires further investigation (Tahara et al., 2002).

Co-expression of hormones
Normally, a cell synthesizes and secretes specific hormones for the regulation of physiological functions and metabolic processes.However, some normal cells and adenoma cells express and secrete many different types of hormones, a phenomenon known as "hormone co-expression" (Radian et al., 2003;Tong et al., 2013;Allensworth-James et al., 2020).Osamura et al. observed the co-expression of GH, α subunits, and gonadotropin in some cells of the anterior pituitary in normal adults (Osamura et al., 2009).This evidence suggests the presence of a certain percentage of plurihormonal cells in the normal anterior pituitary, which refutes the notion of "one cell type, one hormone." Lubov et al. found that the same cells in normal adenohypophysis could co-express PRL with ACTH, TSH, FSH, and LH; GH with ACTH, TSH, FSH, and LH; and TSH with ACTH, FSH, and LH.After comparing 10 scanning chemical sections of the pituitary gland, they found a significant overlap in the GH, PRL, LH, and ACTH expression regions of the distal pituitary gland.Moreover, the co-expression of TSH and PRL was highly prominent in 50% of the anterior pituitary cells using confocal scanning microscopy, with co-expression coefficients ranging from 3.4 to 22%.The co-expression coefficients of PRL and LH were between 4 to15%.The coexpression coefficients of GH and LH ranged from 0.4 to 24%.The coexpression coefficients of ACTH/ PRL and ACTH/GH were 34 and 55%, respectively (Mitrofanova These are the key signaling pathways of pituitary adenomas that may be involved in the initiation and progression of adult pituitary stem cell tumors.The pathways include WNT, TGFβ, NOTCH, HIPPO and SHH.The WNT, TGFβ, and NOTCH pathways may further participate in epitheliummesenchymal transition (EMT), and promote tumor growth and spread.The WNT pathway: The WNT protein binds to the FZD receptor on the cell membrane, leading to the stabilization and accumulation of β-catenin protein, which enters the nucleus and binds to the transcription factor PROP-1, TGF7L2, regulating the expression of the target gene.The TGFβ pathway: TGFβ binds to the receptor complex, phosphorylates to SMAD2/3, and binds to SMAD4 to form a complex that enters the nucleus and binds to transcription factors to regulate the expression of target genes.The NOTCH pathway: After the NOTCH receptor binds to ligand DLL1/JAG1, NICD is released, which enters the nucleus and binds to transcription factor RBP-J to regulate gene expression.The HIPPO pathway: The protein encoded by the Hippo gene interacts with MST to activate the LATS protein, so that YAP and TAZ are phosphorylated and fixed in the nucleus, unable to enter the nucleus to regulate gene expression.When the signaling pathway is suppressed, YAP and TAZ enter the nucleus, bind to the transcription factor TEAD, and regulate gene expression.SHH pathway: In the absence of SHH, the PTCH receptor inhibits the activity of the protein SMO, and when SHH binds to PTCH, PTCH no longer inhibits SMO, thus SMO is activated and GLI protein is regulated.These proteins enter the nucleus to regulate the expression of target genes.EMT: Three families of transcription factors, ZEB, SNAIL, and TWIST, play a central role in EMT.For pituitary stem cell tumors, WNT, TGFβ, and NOTCH pathways may be involved in the regulation of EMT.Cai et al. 10.3389/fnins.2023.1323883Frontiers in Neuroscience 05 frontiersin.orget al., 2017).Therefore, this hormone co-expression phenomenon may also be the cause of PPA.Evidence from immunohistochemistry studies has shown that GH and PRL are co-expressed in the same cell (Kurotani et al., 2001;Tong et al., 2013).Likewise, the cells in pituitary adenomas may display hormone co-expression.Electron microscopy, immunocytochemistry, and laser scanning confocal microscopy revealed that GH and TSH were co-expressed in the anterior pituitary cells of adult rats with methimazole-induced hypothyroidism (Radian et al., 2003).Tong et al. performed immunohistochemical tests on a patient with TSH-GH-PRL multihormone adenoma and found that TSH and GH were colocalized in most tumor cells, indicating that the two hormones were mainly expressed by the same cells.The ultrasonic structure presented a simplex cell appearance and a single type of secretory particle, similar to the secretory particles in normal TSH cells.This suggests that TSH and GH originated from the same secretory particles (Tong et al., 2013).

Differential gene expression
PPA pathogenesis may involve several abnormally expressed genes and pathways.Zhiquan et al. examined the gene expression profiles of seven PPA using a magnetic bead-based optical fiber array, and compared them with those of three normal pituitary glands.A pathway analysis of all differentially expressed genes using the Kyoto Encyclopedia of Genes and Genomes (Jiang et al., 2012) demonstrated that the expression of six genes in PPA increased significantly, and the expression of 334 genes and 15 expression sequence tags decreased significantly.Bioinformatics analysis indicated that HIGD1B, EPS8, ECT2, and BTG2 may play important roles in the tumorigenesis and progression of PPA.Pathway analysis showed that the Notch and p53 signaling pathways may play an important role in the development of PPA tumors.Extracellular matrix (ECM) receptor interactions may play a role in inhibiting tumor invasion and metastasis (Table 1).

Cell transdifferentiation
In the past few decades, the "one cell, one hormone" theory has been widely accepted, and it has been proposed that the phenotype of adenopituitary cells is immutable, and that they are irreversibly committed to producing a single hormone (Kovacs et al., 1989).In recent years, new technologies have emerged to disprove this.Evidence has shown that polyhormone cells may exist in some differentiated cellular pathways (Stefaneanu et al., 1992;Mitrofanova et al., 2017;Asa et al., 2023).Normal gonadotrophs produce gonadotropins, FSH, and LH.Mammosomatotrophs are cells that produce both GH and PRL, and represent a fluid cell type that can transdifferentiate into somatotrophs during growth and lactotrophs during pregnancy (Stefaneanu et al., 1992;Asa et al., 2023).Childs et al. found that a single cell of the pituitary gland in rodents can contain both ACTH and FSH, and explained this observation as "transdifferentiation." One cell type may be transformed into another owing to the genetic and epigenetic factors at play during tumor progression, and are often accompanied by different morphological changes (Vidal et al., 2000).Kovacs et al. reported a case of an ACTH-GH pituitary tumor and identified ACTH in several GH-secreting particles using immunoelectron microscopy.This result suggests that the tumor was originally a GH adenoma that started producing ACTH because of mutations that occurred during tumor progression (Kovacs et al., 1998).Mark et al. reported a case of adenohypophyseal hyperplasia in a patient with primary hypothyroidism, in which TSH and PRL cells proliferated, and GH cells transdifferentiated into cells that secreted both GH and TSH (Jentoft et al., 2012).Sergio et al. found GH-and TSH-labeled doublehormone particles in the cells of TSH-GH adenomas and doublehormone particles in the adenomas of two patients with acromegaly labeled with PRL and TSH, respectively.Using double immunogold labeling in GH-PRL adenomas, they observed that most tumor cells contained dual hormone particles, and some cells contained only GH or PRL particles.These findings strongly suggest that the transdifferentiation of one cell type into another may be the cause of PPA (Vidal et al., 1999).It has been suggested that the interconversion of one cell type into another is not a direct process but occurs through a bihormonal transition cell that contains functional components common to both cells (Frawley and Boockfor, 1991).

Clinical features
Studies have found that approximately 70% of patients with PPA have hormonal symptoms, with acromegaly being the most common (accounting for 50% of cases).Hyperprolactinemia-related symptoms account for 20% of all the cases, whereas gonadotropin-and thyrotropin-related symptoms are rare (Ho et al., 2001).
Ruoyu et al. analyzed the clinical characteristics of 70 patients with PPA and found that these adenomas accounted for 13% of all pituitary adenomas recorded during the same period.53% of the patients had one clinical symptom, 1% had two endocrine symptoms, and none of the patients showed three or more endocrine symptoms.Of the patients, 74.3% had an elevated level of one hormone, 7.1% had an elevated level of two hormones, and only one had an elevated level of all three hormones (Shi et al., 2022).Seckin et al. analyzed the clinical features of 27 patients with PPA, 10 of whom were diagnosed with PAwUIC and 9 with PIT-1 + PPA.The median age was 44.7 years (range, 19-75 years) and 59.3% were males and 40.7% were females.Large adenomas accounted for 88.8%, giant adenomas ≥4 cm accounted for 22.2, 44.4% of patients were accompanied by cavernous sinus infiltration, and nearly half of the patients were highly invasive.77.8 of the patients with PAwUIC, 77.8% showed non-functional pituitary adenomas, and only four patients had hormonal secretory characteristics (Aydin et al., 2019).Mete et al. reported 31 patients with monomorphic multihormone Pit-1 lineage tumors with a mean age of 44.3 years, 11 of whom had hormone secretion symptoms.Most patients have large adenomas, and 30% have tumors that invade the cavernous sinus (Mete et al., 2016).Horvath et al. reported 29 multihormone Pit-1 spectrum tumors in patients with a mean age of 37.5 years, predominantly female (25:4), all of which were macroadenomas, and approximately half of the tumors were aggressive (Horvath et al., 2005).Erickson et al. reported 27 patients with plurihormonal Pit-1 spectrum tumors (mean age, 36.4 years).All tumors were macroadenomas, and 60% of the tumors were aggressive (Erickson et al., 2009)   EPS8, epidermal growth factor receptor pathway substrate 8; ECT2, epithelial cell transforming sequence 2; BTG2, B cell translocation gene 2; COL11A2, alpha2 chain of type XI collagen; ITGB4, integrin beta4; THBS4, thrombospondin 4; COL4A6, alpha 6 chain of type IV collagen; COL11A1, alpha1 chain of type XI collagen; COL6A2, alpha2 chain of type IV collagen; COL6A1, alpha1 chain of type IV collagen; DLK1, the Notch1 inhibitor delta-like 1 homolog; CtBP2, transcriptional corepressor C-terminal binding protein 2; HES1, the hairy enhancer of the split 1; Ep300, histone acetyltransferase p300; GADD45, growth arrest and DNA damage-inducible gene 45; IGFBP3, insulin-like growth factor binding protein 3; PMAIP1, phorbol-12myristate-13-acetate-induced protein 1. 10.3389/fnins.2023.1323883 Frontiers in Neuroscience 07 frontiersin.orgthat PPA co-expressed with Pit-1 and SF-1 in 38 patients (mean age, 53 years; range, 12-79 years).Most tumors had lamellar or nested adherent epithelioid cells that were eosinophilic throughout the intestine.The most commonly secreted hormone is the growth hormone (GH), which causes acromegaly.However, central hyperthyroidism is rare.Furthermore, GATA3 expression was detected in a subset of PPA that expresses both Pit-1 and SF-1 (Asa et al., 2023).
Although PPA immunohistochemistry usually shows a positive expression of multiple hormones, it does not clinically show an increased secretion of multiple hormones.Most patients only show one clinical symptom (Ho et al., 2001), We analyze the possible reasons as follows.(1) Some immunohistochemically positive hormones have fewer positive particles and the hormone content is insufficient (Elhadd et al., 2009;Vora and Karunakaran, 2017).( 2) Although tumor cells can synthesize certain hormones, the secretion mechanism may be obstructed, and when the secretion of one hormone is dominant, the secretion of another may be suppressed (Lamberts and Macleod, 1990;Müller et al., 1999).( 3) Tumors may originate from primitive cells that are not biologically active.Tumor cells can only synthesize hormonal precursors.Although they can cross-react with the corresponding antibody and appear immunopositive, they do not exhibit biological activity (Tordjman et al., 2019).
We have summarized 31 cases of PPA reported in the literature (Table 2).Most patients had aggressive macroadenomas, and the mean age of the patients was 46.51 years, ranging from 13 to 72 years, with 42% men and 58% women.The most common symptom was acromegaly, and most patients opt for surgical tumor removal.
For decades, it was widely believed that pituitary adenomas were monoclonal (Beck-Peccoz et al., 1996;Dahia and Grossman, 1999;Levy and Lightman, 2003).There is much evidence to support the view that pituitary tumors are clonal lesions caused by defects in intrinsic pituitary cells, most of which are based on X chromosome inactivation (Chu et al., 2008;Sav et al., 2012).However, an increasing number of scholars have proposed that multiple synchronous adenomas are of polyclonal origin and that some sporadic tumors may also be polyclonal (Clayton and Farrell, 2001;Asa and Ezzat, 2002;Buch et al., 2002).PPA can potentially be explained also by heteroclonality (Herman et al., 1990;Levy and Lightman, 2003).In an earlier study based on nuclear DNA analysis by flow cytofluorimetry, biclonal cell lines were isolated in 10% of pituitary adenomas (Anniko and Tribukait, 1985).In more than half of the "recurrent" pituitary tumors with repeated analysis of loss of heterozygosity after further surgery, the loci involved in the original pattern of loss of heterozygosity were heterozygous again (Clayton et al., 2000).It also suggests that these pituitary tumors may be polyclonal.In addition, a growing number of studies have found that pituitary tumors can display intrinsic heterogeneity and cellular subpopulations with different biological, genetic and epigenetic properties (Sabatino et al., 2022).Tissue structure and microenvironment play a crucial role in tumors.Different cellular processes alter the structure and interactions within the gland, thereby shaping tumor growth.Tumor heterogeneity exists in many forms, ranging from somatic coding and non-coding alterations to epigenetic, transcriptomic and post-translational modifications (Caswell and Swanton, 2017;Mcgranahan and Swanton, 2017).

Treatment
PPA is characterized by its aggressiveness and high postoperative recurrence rate (Steinbichler et al., 2018;Lodge et al., 2019;Lan and Behrens, 2023).Transsphenoidal microsurgical resection of mixed pituitary adenomas remains the preferred treatment for PPA (Swearingen, 2012;Ammirati et al., 2013).Surgical removal of tumors can reduce tissue pressure and hormone levels.However, it is not ideal for controlling tumor development for long-term remission.Radiotherapy is used only in patients whose tumor size or hormone levels are not sufficiently reduced by surgery or medication.Radiotherapy can be used as a postoperative adjuvant therapy.Importantly, the monitoring of pituitary function indefinitely after radiotherapy to detect anterior pituitary hypofunction is recommended.Approximately 50% of patients develop hypopituitarism within five years of radiation therapy (Loeffler and Shih, 2011;Ding et al., 2014;Molitch, 2017;Tritos and Miller, 2023).Wang et al. reported a case of GH-PRL adenoma in which programmed death ligand 1 (PD-L1) protein and CD8+ lymphocyte infiltrations were detected in tumor tissue.The tumor was aggressive and did not respond to bromocriptine therapy.GH and PRL levels did not improve after surgical resection and recurred postoperatively.The authors proposed the use of PD-1 inhibitors in combination with radiotherapy or temozolomide for the treatment of aggressive PPA (Wang et al., 2017) John et al. also found a higher incidence of significant PD-L1 in PIT-1positive multihormone tumors than in other pituitary adenomas, and PD-L1 overexpression was very rare in ACTH and gonadotropin cells.The authors suggested that immunosuppressants may be a reasonable treatment for PIT-1 positive plurihormonal adenoma (Turchini et al., 2021).

Conclusion
PPA is a relatively rare tumor that can secrete more than two hormones that differ in their biochemical composition, immunohistochemistry, and biological effects.We conducted a review of the literature to elucidate the clinical features of PPA and suggested the potential pathogenesis.We hypothesized that the pathogenesis of PPA is related to plurihormonal primordial stem cells, co-transcription factors, hormone co-expression, differential gene expression, and cell transdifferentiation.In the future, more basic and clinical studies are required to verify the pathogenesis and clinical features of PPA.

Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article.This work was supported by grants from the National Natural Science Foundation of China (81970687 and 82270850 to GW), the Department of Science and Technology of Jilin Province (20210303001SF, 20190901006JC and YDZJ202202CXJD042 to GW, 20210101439JC to XZ), and the first hospital of Jilin University (JDYYZH-2102030 to GW, JDYYCB-2023010 and 2021-zl-01 to XZ).

FIGURE 1
FIGURE 1The pituitary gland consists of the anterior lobe (AL), intermediate lobe (IL), and posterior lobe (PL).Plurihormonal primordial stem cells (PSC) are thought to reside in the intermediate lobe (IL) and the dorsal anterior lobe (AL), and are dispersed throughout the AL parenchyma.PSCs proliferate into adenomas under conditions that activate oncogenes, inactive tumor suppressor genes, dysregulate cell cycles, or cause mutations.Under the regulation of different transcription factors, the PSCs differentiate into different cells and secrete a variety of hormones.
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TABLE 1
Genes and pathways that may be involved in abnormal expression in PPA.

TABLE 2
Published reports on plurihormonal pituitary adenomas.