Pleiomorphism plurihormonal Pit-1-positive macroadenoma with central hyperthyroidism: a rare case report and literature review

Background: Thyrotropin-secreting pituitary neuroendocrine tumors (PitNETs) are rare pituitary adenomas that are occasionally accompanied by hypersecretion of other anterior pituitary hormones, such as growth hormone (GH) and prolactin (PRL). The clinical, biochemical, and pathological characteristics may represent diverse circumstances. Case presentation: In this report, a 33-year-old female diagnosed with a TSH PitNET co-secreting GH presented no obvious clinical symptoms. The main characteristics were elevated thyroid-stimulating hormone (TSH), free tri-iodothyronine (FT3), and free thyroxine (FT4) levels accompanied by slightly elevated GH and insulin-like growth factor-1 (IGF-1) levels. Magnetic resonance imaging (MRI) detected a pituitary macroadenoma (18 × 16 × 16 mm) with cavernous sinus and suprasellar invasion. Immunohistochemistry revealed diffuse positivity for TSH, strong immunoreactivity for GH, and sporadic positivity for PRL. The electron microscope and double immunofluorescence staining confirmed a plurimorphous plurihormonal adenoma producing TSH, GH, and PRL. After preoperative somatostatin receptor ligand (SRL) treatment and transsphenoidal surgery, the patient achieved temporary clinical and biochemical remission. However, 3 months after surgery, the patient was suspected of having Hashimoto’s thyroiditis due to higher thyroglobulin antibody (TGAb), thyroid peroxidase antibody (TPOAb), and thyroid receptor antibody (TRAb) and an enlarged thyroid nodule. During follow-up, thyroid function and TSH slowly transformed from transient hyperthyroidism to hypothyroidism. They were maintained in the normal range by L-T4. Conclusion: In the TSH PitNET, the positive immunohistochemistry for TSH, GH, and PRL translated into hormonal overproduction with TSH and GH


Background
Thyrotropin-secreting pituitary neuroendocrine tumor (PitNET) with a prevalence of 2-3 cases per million is an infrequent cause of central hyperthyroidism and is estimated to be less than 3% of all PitNETs [1,2]. The chief characteristics of TSH PitNETs are inappropriate thyroid-stimulating hormone (TSH) levels (normal or high) and elevated free thyroxine (FT4) and free tri-iodothyronine (FT3) levels. Some patients with a long history of thyroid dysfunction tend to be easily misdiagnosed with Graves' disease. Pit-1 is a common critical terminal differentiation factor and co-secretes in thyrotrophs, somatotrophs, and lactotrophs. One-fourth of TSH PitNETs are plurihormonal adenomas of Pit-1 lineage with hypersecretion of other pituitary hormones, mainly growth hormone (GH) (18%) and prolactin (PRL) (9%) [2].
To our knowledge, the frequency of pituitary adenomas producing TSH, GH, and PRL is relatively rare; only a few cases have been reported [3][4][5][6][7][8][9][10][11]. According to the 2017 World Health Organization (WHO) classification of tumors of the pituitary gland [12], a completely new definition is the plurihormonal Pit-1-positive adenoma, previously called silent adenoma subtype 3. Most of these tumors are clinically silent and rarely function as adenomas with acromegaly, hyperthyroidism, or hyperprolactinemia [13]. Additionally, as they are atypical lesions on conventional histology and immunohistochemistry, a definitive diagnosis usually requires ultrastructural confirmation. To provide more information for the clinician, this review focuses on the clinical, pathological, and therapeutic findings of a plurihormonal Pit-1-positive macroadenoma presenting with central hyperthyroidism.

Case presentation
A 35-year-old female was referred to our department in October 2021 because of thyroid dysfunction for 2 months. Her past and family histories were unremarkable. In the local hospital, thyroid function tests demonstrated high levels of free tri-iodothyronine (FT3) 10.18 pmol/L (3.1-6.89 pmol/L), free thyroxine (FT4) 25.8 pmol/L (11-22 pmol/L), and thyroid-stimulating hormone (TSH) 8.6 mU/L (0.27-4.2 mIU/L). Thyroid ultrasound revealed a solid, slightly hyperechoic nodule in the left lobe of the thyroid (9.6 × 7.8 × 10.1 mm). Based on this funding, the patient was diagnosed with Graves' disease and given methimazole (MMI, 5 mg bid). After 20 days, her thyroid function test was rechecked: FT3 was 7.97 pmol/L, FT4 was 19.8 pmol/L, and TSH was 10.49 mU/L. A local hospital suspected the patient had TSHoma. Further inquiries did not find a family history of thyroid dysfunction.
On examination at our hospital, her body mass index (BMI) was 22.8 kg/m 2 , her blood pressure and heart rate were 116/79 mmHg and 105 bpm, respectively, and her body temperature was 37 °C. Physical examination showed a degree 2 enlarged thyroid gland with no oculopathy or symptoms of acromegaly (Supplementary Figure 1).

Preoperative imaging examination
Her initial brain magnetic resonance imaging (MRI) revealed a quasi-circular equal signal shadow in the sellar region and a pituitary macroadenoma (18 × 16 × 16 mm) adjacent to the siphon of the left internal carotid artery (Fig. 2). The T2-weighted signal intensity was isointense. In addition, the Knosp and Hardy classifications of the pituitary tumor were grade 1 and 2, respectively. Additionally, the cavernous sinus invasion score (CSIS) was grade 1, the sphenoid sinus invasion score (SSIS) was grade 0, the suprasellar extension score (SSES) was grade 1, and the cumulative score was grade 2 [14]. The visual field revealed a visual field defect in the left eye. In thyroid echography, enlarged thyroid and hyperechoic nodules in the left lobe of the thyroid (11 × 8.3 × 11 mm, TI-RADS class 3) were observed. An ultrasound-guided fine-needle biopsy of the thyroid nodules showed benign lesions.

Diagnosis and treatment
Based on these findings, this patient was finally diagnosed with TSH PitNET and acromegaly. After our multidisciplinary united team consultation, the patient was preoperatively treated with octreotide (OCT, 0.1 mg, s.c., tid). TSH, FT3, and FT4 levels decreased to the normal range in 5 days. Then, the pituitary mass was endoscopically removed via an endoscopic transsphenoidal resection. Surgical pathology confirmed a macroadenoma. Histologically, the tumor was composed of plurimorphic cells with a distinct cell border, abundant granulated cytoplasm, and round or oval nuclei. On light microscopy, histopathology revealed the plurihormonal adenoma with strong nuclear immunoreactivity with Pit-1 antibody (Fig. 3a). Immunohistochemistry revealed strong immunoreactivity for GH (Fig. 3b) and diffuse positivity for TSH (Fig. 3c) and PRL (Fig. 3d). The tumor had negative staining for ACTH, FSH, LH, and ER. In p53 staining, scattered p53-positive cells were observed (Fig. 3f ). The Ki-67 index was < 1% (Fig. 3e). In GH and TSH immunohistochemical double staining, many GH cells and a minority of TSH cells were observed (Supplementary Figure 3B). Immunofluorescence double staining for GH and TSH positivity was found in different cell populations (Supplementary Figure 3A). In GH and PRL immunohistochemical and immunofluorescence double staining, GH was the most diffusely positive hormone, and PRL reactivity was scattered (Supplementary Figure 3C, D). Electron microscopy revealed that the tumor consisted of plurimorphous cells (Fig. 4). Some adenoma cells (black arrow) were densely granulated. Secretory granules were spherical or ovoid and measured 300-450 nm. Many adenoma cells contained elongated or geometrically shaped secretory granules, suggesting crystallization within their substance, a phenomenon seen in densely granulated somatotropic adenomas. The thyrotropin cells (red arrow) had a predominantly spherical or ovoid nucleus, scattered lysosomes and mitochondria, Fig. 1 The suppression ratio of TSH and GH during 24 hours octreotide tests. The maximum suppression rate of GH was 94.4% at 2 hours; The maximum suppression ratio of TSH was 81.2% at 24 hours prominent Golgi apparatus, and numerous secretory granules measuring 100 to 200 nm. The cell membrane was peripherally clustered with numerous small secretory granules that outlined the cell boundary.
After surgery, TSH decreased slightly below the normal range (0.299 mIU/l), and FT3 and FT4 levels were normal at 2.75 pmol/L and 9.9 pmol/L, respectively. GH and IGF-1 levels were 0.16 μg/mL and 274 ng/mL 1 week after the surgery (Table 1). Except for high TGAb and TPOAb levels, TSH, FT3, FT4, GH, and IGF-1 levels decreased to the normal range 1 month after surgery. The OGTT test successfully suppressed GH below 0.4 μg/mL (basal GH, 7.62 μg/mL; maximal suppression, 0.4 μg/mL, Supplementary Figure 2). However, although no clinical symptoms of hyperthyroidism were observed in this patient, FT3 and FT4 levels increased, and TSH levels were slightly below the normal range 3 months after surgery (Table 1). Considering previously consistently elevated TGAb, TPOAb, and TRAb levels and an enlarged thyroid nodule, the patient was suspected of having Hashimoto's thyroiditis. Interestingly, her thyroid function normalized over 10 days without medical treatment (Table 1). Four months after the surgery, her thyroid function showed increased TSH, TRAb, TPOAb, and TGAb levels and decreased FT4 levels. Hashimoto's thyroiditis further destroyed the thyroid cells and led to hypothyroidism in this patient. Indeed, central hypothyroidism was also possible. Six months after the surgery, the patient was given levothyroxine sodium tablets (L-T4, 37.5 μg/day), and her FT4, TSH, and TRAb returned to the normal  Table 1). The MRI revealed no particular abnormalities after the surgery. The patient achieved basal clinical and biochemical remission during follow-up.
Immunocytochemical staining showed that this TSH PitNET was a plurihormonal tumor of Pit-1 lineage producing TSH, GH, and PRL. Concurrently, electron microscopy confirmed that TSH and GH were secreted by two different cells. Interestingly, although the pathology confirmed scattered PRL expression, the hypersecretion of PRL was not found in the blood. Previous cases also described plurimorphous plurihormonal adenomas producing TSH, GH, and PRL [17,18]. However, in these reports, no fluorescent immunostaining studies had been administered to confirm the co-secretion of various hormones. In addition, Ozawa et al. reported that a plurihormonal adenoma co-secreting TSH and GH was ultrastructurally monomorphous in electron microscopy but immunohistochemically polymorphous [11]. Several reports confirmed that most plurihormonal TSHomas were monomorphous [4,5,8]. The plurihormonal Pit-1-positive tumors were characterized by a Fig. 4 Electron microscopy revealed the adenoma cells were Plurimorphous. Ultrastructural examination identified that some adenoma cells were densely granulated, and secretory granules were spherical or ovoid and measured 300-450 nm (black arrow). The thyrotropin cells were a predominantly spherical or ovoid nucleus, Scattered lysosomes and mitochondria, prominent Golgi apparatus, and numerous secretory granules measuring 100 to 200 nm (red arrow) monomorphous population of cells expressing more than one hormone: TSH, GH, PRL, and α-subunit.
High-resolution pituitary MRI as a noninvasive technique can better characterize pituitary adenoma, predict postoperative pathology, and guide clinical treatment. The plurihormonal Pit-1-positive adenomas were highly aggressive and invasive, especially predominant suprasellar adenomas [19]. A previous case reported that a brain MRI in a pediatric patient revealed a giant plurihormonal pituitary adenoma with highly suprasellar extension, sphenoid, and bilateral cavernous sinus invasion (Knosp score 3) [5]. Differently, the tumor in our patient was typically macroadenoma with slightly cavernous and sphenoid sinus invasion (Knosp score 1). Additionally, T2 intensity indicated a significant relationship between histological subtype and the therapeutic effect of somatostatin receptor ligands (SRLs). T2 hyperintense GHsecreting adenomas were similar to sparsely granulated adenomas accompanied by typically higher cavernous sinus invasion and had a blunted response to first-generation SRLs [20]. Recently, studies reported that T2-signal intensity might change during SRL treatment, especially second-generation SRLs [21,22]. In our case, immunohistochemistry demonstrated that GH cells had the characteristics of dense granules in electron microscopy. This tumor was isointense on T2-weighted magnetic imaging. The patient presented with hyperthyroidism and acromegaly received octreotide treatment, and obtained apparent clinical and biochemical control.
This case report had some limitations. First, the follow-up of this patient did not exceed 1 year. Second, this case performed no further colloidal gold double staining under the electron microscope. Third, the lack of somatostatin receptors 2 and 5 stainings could not help guide and evaluate the efficacy of SRLs.

Conclusions
This case presented atypical clinical symptoms and hypersecretion of TSH and GH with normal PRL levels. However, the histology and ultrastructure proved that this pituitary adenoma had three distinct cells producing TSH, GH, and PRL. Concurrently, due to the combination of TSH PitNET and Hashimoto's thyroiditis in this patient, dynamic testing of thyroid function (TSH, FT3, and FT4) and corresponding antibodies (TRAb, TPOAb, TG, and TGAb) helped to diagnose and identify the cause of thyroid function variations and guided followup treatment. Preoperative octreotide therapy combined with transsphenoidal surgery as the treatment for this patient ensured the safety of the perioperative period and achieved an excellent therapeutic effect.