Growth hormone-releasing hormone-secreting pulmonary neuroendocrine tumor associated with pituitary hyperplasia and somatotropinoma

SUMMARY Acromegaly caused by ectopic growth hormone-releasing hormone (GHRH)-secreting tumor is exceedingly rare. We report a case of acromegaly secondary to GHRH secretion by an incidentally diagnosed pulmonary neuroendocrine tumor (NET) and review 47 similar cases in literature. A 22-year-old male patient presented with symptoms of pituitary apoplexy. Magnetic resonance imaging (MRI) showed apoplexy of a pituitary adenoma. Routinely prior to surgery, a chest radiography was performed which revealed a mass in the left lung. During investigation, the patient was diagnosed with metastatic GHRH-secreting pulmonary NET. In retrospect, it was noted that the patient had pituitary hyperplasia 20 months prior to the MRI which showed the presence of a pituitary adenoma. The histological findings confirmed somatotroph hyperplasia adjacent to somatotropinoma. This case suggests that GHRH secretion can be associated with pituitary hyperplasia, which may be followed by pituitary adenoma formation.


INTRODUCTION
A cromegaly is a chronic disease due to excess growth hormone (GH) and insulin-like growth factor type I (IGF-I) (1).GH-secreting pituitary adenomas (somatotropinomas) are responsible for the vast majority of cases, while ectopic GHreleasing hormone (GHRH) secretion is exceedingly rare accounting for less than 1% of cases (2).In the context of ectopic secretion, acromegaly is caused by pulmonary neuroendocrine tumors (NET) in majority of cases, followed by gastro-enteropancreatic NET, and rarely by pheochromocytomas or paragangliomas (3).Pulmonary NET are uncommon, having an incidence of 0.2 to 2 per 100,000, and are classified as typical carcinoid, atypical carcinoid, large cell neuroendocrine carcinoma and small cell neuroendocrine carcinoma based on 2015 World Health Organization (WHO) classification (3,4).About 70 cases of ectopic secreting GHRH tumors have been described to date with pulmonary NETs representing the majority of cases (5).
The clinical features of ectopic acromegaly are indistinguishable from those resulting from GHRH-pulmonary NET and somatotropinoma Arch Endocrinol Metab.2021;65/5 somatotropinoma (5).However, some findings are suggestive of ectopic secretion such as pituitary hyperplasia which is related to prolonged GHRH hypersecretion, local tumoral symptoms (from airway obstruction in the case of pulmonary NETs), cosecretion of humoral factors synthetized by welldifferentiated NETs causing carcinoid syndrome (6,7).Excessive circulating serotonin levels leads to carcinoid heart disease, diarrhea and abdominal pain (7).
We report a case of a male patient who was diagnosed with acromegaly due to GHRH-secreting pulmonary NET and somatotropinoma, who presented with carcinoid syndrome, and review cases of GHRHsecreting pulmonary NETs published in literature.
This study was conducted in accordance with the ethical standards of the Helsinki Declaration, with the approval of the Instituto Estadual do Cérebro Paulo Niemeyer Ethics Committees involved in the study (CAAE 39632820.8.0000.8110).Patient's written consent was obtained for publication.

CLINICAL SUMMARY Clinical presentation
A 22-year-old male patient presented with sudden and severe headache after dental extraction.Brain computed tomography (CT) showed an isodense pituitary lesion, with no contrast enhancement.No visual disturbance was present.Sellar region magnetic resonance imaging (MRI) performed nine days after the headache revealed a 3.3 x 2.4 x 1.4 cm lesion suggestive of pituitary apoplexy (PA) (Figure 1).The patient exhibited mild acromegaly features with mandibular prognathism, prominent forehead and tall stature that was familial.
Of note, the patient had a brain MRI performed 20 months before due to mental confusion and dizziness that showed slight enlargement of pituitary gland of 9.31 mm (normal range adjusted for sex and age: 5.63 + SDS 1.00) (8).The patient also complained of occasional abdominal cramps and diarrhea that started one year before and had an unexplained weight loss of 10 kg during the last two months.The patient had no family history of endocrine tumor.
On pre-operative evaluation, a routine chest X-ray was performed that showed a 4 cm central lesion in inferior left pulmonary lobe (ILL).CT scan demonstrated a 4.5 x 4.0 x 3.5 cm lesion in ILL. 18Fluorfluorodeoxyglucose positron emission tomography ( 18 F-FDG PET) CT scan and 68 Gallium DOTA-Tyr3octreotide ( 68 Ga-DOTATOC) PET CT revealed discrete increase in glycolytic metabolism and significant increase of somatostatin receptor expression in the ILL, left lobe hilum, hepatic hypovascular lesions, spine, sacrum and pelvis, consistent with a well differentiated metastatic NET (Figure 2).A bronchoscopy showed a vegetative and hypervascularized lesion, obstructing the left bronchial segment.The bronchial lesion was biopsied and was compatible with atypical pulmonary

Pulmonary tissue analysis
RNA from the FFPE tissue was extracted from the pulmonary specimen, at a concentration of 13.6 ng/uL.Reverse transcription (RT) and quantitative polymerase chain reaction (qPCR) were performed as previously reported (10), showing very high expression levels of GHRH mRNA in the pulmonary NET (5053 mRNA copies) compared to six normal pulmonary tissues (Figure 4).Three reference genes [beta-actin (ACTB), hypoxanthine guanine phosphoribosyl transferase (HPRT) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] were also measured being ACTB the most stable control gene; therefore, ACTB was used to control for variations in the amount of RNA used in the RT reaction and to adjust the relative expression of each of the transcripts analyzed.GH was not expressed in the sample.The specific primers used in qPCR are detailed in Sup.Table 1.At protein level, anti-GHRH (dilution 1:750, Abcam, cat.number ab187512) was also positive. of serum IGF-I to 197 ng/mL (99-289) and urine 5-hydroyindoleacetic acid to 8.9 mg/24h (2.0-9.0) were observed.Serum chromogranin A and serotonin levels decreased significantly but did not reach normal values [470 ng/mL (25-140) and 257.7 mcg/L , respectively].Brain natriuretic peptide levels and echocardiography are normal.

Genetic sequencing
Multiple endocrine neoplasia type 1 (MEN1) and 4 (MEN4) were investigated.Peripheral blood samples were collected with EDTA as anticoagulant and DNA was extracted from leukocytes.Somatic mutations were not analyzed due to paucity of tumor samples.Gentra PureGene Blood Kit (Qiagen, Minnneapolis, MN, USA) was used to obtain genomic DNA.The eluted was resuspended in 100 μL of DNA Hydration Solution following the manufacturer's instructions.After extraction, PCR reactions were performed on Applied Biosystem ProFlex™ PCR System (Thermofisher, Foster City, CA, USA).Amplification and sequencing were performed with PCR/Sanger Sequencing Primer pairs (ThermoFisher Scientific™, Boston, MA).Products were sequenced in both directions on ABI 3130xl Genetic Analyzer (Applied Biosystems) and electropherogram-derived sequences were aligned by using Benchiling and BioEdit software (Sup.Table 2).Germline MEN1 and cyclin dependent kinase inhibitor 1B (CDKN1B) sequencing were negative for pathogenic mutations.

DISCUSSION
The patient was initially diagnosed with pituitary adenoma (PA) and afterwards with pulmonary mass which turned out to be a GHRH-secreting pulmonary NET associated with somatotropinoma and pituitary hyperplasia.Because of extensive metastasis, primary pulmonary tumor was not resected and pegvisomant and octreotide LAR were started early, followed by PRRT.Compared to literature, our case is the only case in which PA and pituitary hyperplasia were observed in pathology, and pituitary hyperplasia was seen on MRI scans before pituitary adenoma formation.
To our knowledge, only 47 cases have been described of GHRH-secreting pulmonary NET causing acromegaly (48 including our case -Sup.Table 3).Literature review using PubMed and Google Scholar databases was performed with the following keywords:

Medication
Levothyroxine, prednisone and testosterone were prescribed for hypopituitarism.Pegvisomant 70 mg/week and octreotide LAR 20 mg/month were started after ectopic acromegaly secondary to GHRHproducing metastatic tumor was diagnosed, while waiting for peptide receptor radionuclide therapy (PRRT).Octreotide LAR was titrated to 30mg and was given after 177 Lu dose every 6 weeks.After four cycles of PRRT, 18 F-FDG PET scan revealed complete metabolic resolution of the lesions and reduction of the primary pulmonary NET in about 22% in axial view.Pegvisomant was titrated to maintain IGF-I levels in normal range.Nowadays the patient is on pegvisomant 40 mg/week and octreotide LAR 30 mg/month.After one year of therapy, normalization

GHRH-pulmonary NET and somatotropinoma
Arch Endocrinol Metab.2021;65/5 "GHRH-secreting tumor", "bronchial carcinoid", "pulmonary neuroendocrine tumor" and "ectopic acromegaly".From literature review (Sup.Table 3), GHRH-secreting pulmonary NET was more frequent in females representing 70.8 % of cases (34/48), which was not our case.Median age of the patients was 42.5 years (range: 19-77 years), older than our patient.GHRH-secreting pulmonary NET were usually large tumors in most cases with a median diameter of 4.5 cm (1.2-9) on chest CT, like our case, and were more common on the right side [19 of 28 cases (67.8%) in which side was described], which was not our case.NETs were diagnosed by histology with the presence of positive immunostain for chromogranin and GHRH in the pulmonary specimen as found in our case, or suspected with positive functional imaging with increased serum GHRH.All patients except for three were submitted to pulmonary resection, even in the presence of metastatic lesions (11,12).Data on subtype of NET was available in 18 patients: 10 (55.5%) had typical pulmonary carcinoids and eight (44.5%) had atypical tumors.
In line with our case, 14 (33.3%)patients already had distant metastases at diagnosis.Most common sites were liver and/or bone present in 12 (75%) of these metastatic cases, similar to our case.Other sites included lymph nodes, breast, ovaries, heart, thyroid gland and even one case of metastasis to pituitary gland (13).
As NET can secrete bioactive amines such as serotonin, patients can present with carcinoid syndrome.This syndrome was uncommon in pulmonary NET (8% of patients) because of lower production of serotonin compared to other NET, but can be more frequently encountered in patients with liver metastases (15% of patients), as was our case (14).A delay of several years frequently occurred before a diagnosis of carcinoid tumor was made (15).
Pulmonary NET can also secrete GHRH causing acromegaly.An association may exist between ectopic acromegaly and genetic syndromes.Pulmonary NET has been reported in the context of MEN1 and MEN4 (16).Approximately 200 somatic MEN1 mutations have been described in several different endocrine tumors, with 26 cases being of pulmonary NET (17)(18)(19)(20).Although acromegaly can be seen in MEN1, in these pulmonary NET series it was not present.With respect to MEN4, one case of germline CDKN1B mutation who had pulmonary NET and pituitary adenoma has been described, but it was not of somatotroph origin (21).
Acromegaly was usually diagnosed before pulmonary NET as described in 33 patients of 48 cases (68.7%).Patients had signs and symptoms of acromegaly (prognathism, acral enlargement, hyperhidrosis) for median period of 8.5 years (2-26) prior to diagnosis.In these cases, patients had normal sellar imaging or findings suggestive of pituitary hyperplasia, so ectopic secretion was investigated.Interestingly, 12 patients already had a history of pulmonary mass or resected pulmonary tumor.Only five patients (10.4%) had local symptoms (pneumonitis or hemoptysis) and only four patients (8.3%) carcinoid syndrome.In five cases (10.4%), incidentally discovered pulmonary mass was seen on routine chest X-ray prior to surgery, as in our case.
In our case, pituitary hyperplasia was seen before adenoma formation, which has not been described previously.In the other cases, pituitary hyperplasia was seen during acromegaly investigation.It was present in 50% of patients who were submitted to brain or sellar imaging (CT or MRI).Eight (20%) cases were suspected of having pituitary tumor, but only five cases confirmed pituitary adenoma at histopathology examination (four somatotropinoma and one null cell adenoma) (5,13,22,23).Another patient might also have had an acidophilic cell adenoma or hyperplasia (indistinguishable) (24).In other five cases, patients were operated for a presumed somatotropinoma but histological examination only revealed pituitary hyperplasia (intact reticulin fiber network), suggesting that differentiation between pituitary hyperplasia and adenoma based on imaging was very difficult.
Pituitary adenoma and hyperplasia can coexist as we have seen in our case.In some reports, adenomatous transformation from pituitary hyperplasia have been described (5).In the five cases with pituitary adenoma, patients presented with acromegaly at earlier age [23 years old (19-44)] and was more common in men (3/5 cases), as seen in our case.Pulmonary NET was diagnosed incidentally in two cases including our own, or during investigation of metastatic tumor or of uncontrolled acromegaly (13,22,23).Apart from our case whose initial symptom was pituitary apoplexy, in the other cases, pulmonary NETs were present for at least 7.5 years (2-9) before a pituitary adenoma could be seen, which makes our case even more intriguing.Pituitary hyperplasia was observed on MRI before adenoma formation, which occurred relatively fast (for at least 20 months).We also presume our patient developed pituitary tumor rapidly because he presented GHRH-pulmonary NET and somatotropinoma Arch Endocrinol Metab.2021;65/5 with pituitary apoplexy.Rapid growth can precipitate apoplexy because tumoral blood vessels have inherent fragility contributing to hemorrhagic tendency (25).Additionally, another unique finding in our case was that of pituitary hyperplasia adjacent to adenomatous tissue on histopathology, rather than a mixture of hyperplasia and adenoma formation or a transition zone as seen in literature.
Biermasz and cols.( 5) described a 27-year-old patient that had pituitary macroadenoma concomitant with GHRH-secreting pulmonary NET and had histopathological characteristics of a mixture of pituitary hyperplasia and adenoma formation in pituitary specimen.Also, Nasr and cols.(13) reported a case in which metastatic GHRH pulmonary NET to the pituitary was present adjacent to somatotroph hyperplasia and focal neoplastic transformation of sparsely granulated somatotroph phenotype suggesting a relation between GHRH hypersecretion and somatotroph cell disarrangement.
Similarly, in gangliocytoma (hypothalamic GHRHproducing tumor), somatotropinomas could be concomitantly seen (26,27).Moreover, in GHRH transgenic mice, histological findings resembled that of humans case reports in which diffuse pituitary hyperplasia and somatotropinoma can be found (28,29).Billestrup and cols.(30) reported that in anterior pituitary rat cells marked for GH, GHRH exposure lead to higher mitogenic index and GH secretion in these cells.These results were also seen when cells were treated with forskolin, which stimulates cyclic adenosine monophosphate (cAMP) production, mimicking GHRH receptor (GHRH-R) activation by its second messenger.Kineman and cols.( 31) also reported in their study in mice that GHRH stimulation causes hypertrophic and hyperplastic effect in somatotrophs, also having found characteristic features of pituitary adenoma.Nevertheless, they believe the onset of pituitary adenomas was not preceded by a progressive increase in pituitary size, however these authors presume that when protective mechanisms are lost due to hyperactivation of GHRH-R in selected cells, adenoma formation occurs (31).Luque and cols.(32) showed that constitutive activation of GHRH-R in transgenic mice was associated with pituitary hyperplasia and adenoma formation late in life.These studies suggested that excess circulating GHRH could lead to pituitary hyperplasia and adenoma formation in animal pituitary.
In humans, a causative association between GHRH hyperstimulation and pituitary somatotroph adenoma formation has not been demonstrated.Theoretically, there may be neoplastic transformation preceded by pituitary hyperplasia.Ectopic GHRH excess usually results in pituitary hyperplasia alone (Sup.Table 3) that has been described to be reversible (33).A hypothesis proposed is that the pituitary gland responds to trophic hormone secretion by undergoing reversible plastic changes in cell proliferation (34).Increased cell proliferation would lead to reversible hyperplasia or to pituitary adenoma formation depending on additional secondary genetic events.These events would be required for adenoma formation since not all cases develop pituitary adenoma (32,34,35).
Interestingly, other cases of somatotropinomas can also be associated with pituitary hyperplasia, as Villa and cols.(36) reported that tumors of non-identical twin sisters with AIP mutation had transition zones from hyperplasia to adenoma.Our case, however, did not show a transition zone from hyperplasia to adenoma in the pathological analysis, rather it showed pituitary adenoma adjacent to pituitary hyperplasia.
Regarding treatment, for acromegaly, pegvisomant is the drug with the highest efficacy and therefore was prescribed from the beginning for optimal and rapid IGF-I lowering (37,38).For pulmonary NET, surgical resection can cure the disease, however, in advanced metastatic cases, other options should be considered.Optimal management in the setting of advanced unresectable disease is not well established.Somatostatin receptor ligands (SRL) have been used in the treatment of ectopic acromegaly as they interrupt the pathophysiological process generated by ectopic GHRH secretion through suppression of tumoral GHRH and inhibition of GH secretion from the anterior pituitary gland (39).However, despite the favorable clinical improvement documented for some acromegaly patients, the response of GHRH-secreting tumor and its metastases is less predictable.For disseminated disease, such as our case, systemic therapy with radiolabeled somatostatin analogue therapy (also known as PRRT) can be appropriate.Lutetium has been studied mainly in gastroenteropancreatic NET in the NETTER-1 trial and in some series of pulmonary NET (40,41).Nevertheless, in a cost-effective, more economic approach, high doses of SRL (42) can be considered as monotherapy initially for NET with pegvisomant being prescribed after failure of SRL and PRRT.

GHRH-pulmonary NET and somatotropinoma
Arch Endocrinol Metab.2021;65/5 In conclusion, very few cases of acromegaly secondary to GHRH-secreting pulmonary NET have been described, and even fewer of associated somatotropinoma.Despite its rarity, pituitary adenoma may coexist with pituitary hyperplasia in acromegaly due to ectopic origin.In the setting of acromegaly, ectopic GHRH or GH secretion must be thought of when pituitary scan shows pituitary hyperplasia or does not reveal a lesion suspicious of adenoma.Although generalizations cannot be made by case reports, we speculate, in our case, that the GHRHproducing pulmonary NET might have led to the development of pituitary hyperplasia and posteriorly to somatotropinoma formation that was manifested as PA.

Figure 1 .
Figure 1.MRI at 22 years of age in coronal T1W (a), post-contrast T1W (b), coronal T2W (c) and sagittal T1W (d) views showing a 3.3 x 2.4 x 1.4 cm sellar lesion, with suprasellar extension and optic chiasm compression, heterogeneous, predominantly isointense in T1W and T2W, with peripheral contrast enhancement, exhibiting an internal small area with hypointense rim in T2W, suggestive of pituitary adenoma with hemorrhagic degeneration in different stages of evolution.

Figure 3 .
Figure 3. Histological sections showing enlarged lobules of the adenohypophysis (pituitary hyperplasia) in HE (a); pituitary hyperplasia and adenoma (*) in HE (b); GH positive in PA and pituitary hyperplasia (c); prolactin positive in pituitary hyperplasia (d); ACTH positive in pituitary hyperplasia (e).

Table 2 .
Disclosure: MRG has received speaker fees from Novartis, Ipsen and Pfizer and attended advisory boards for Novartis and Crinetics.LK has received speaker fees from Novartis and Ipsen.EBL, DGH, MCVB, CHAL, RML, LC have no conflict of interest.No sponsor has been involved in this study.Genetic sequencing Codons of interest were amplified in 25 μL PCR reaction mixture.The amplified products were then visualized on 2% agarose gel stained with SYBR™ Safe DNA Gel Stain (Invitrogen by ThermoFisher Scientific).PCR products were purified from unincorporated nucleotides and primers using the ExoSAP-IT™ PCR Product Cleanup Reagent (Affymetrix Inc. by ThermoFisher Scientific).Direct sequencing of purified PCR products were performed in both directions on 3130XL Genetic Analyzer automatic sequencer (Applied Biosystems by ThermoFisher Scientific) and analyzed using the Benchiling software (https://benchling.com/)and BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).

Table 3 .
Literature review