Complicated Clinical Course in Incipient Gigantism Due to Treatment-resistant Aryl Hydrocarbon Receptor–Interacting Protein–mutated Pediatric Somatotropinoma

Background Our objective was to describe the clinical course and treatment challenges in a very young patient with a pituitary adenoma due to a novel aryl hydrocarbon receptor–interacting protein (AIP) gene mutation, highlighting the limitations of somatostatin receptor immunohistochemistry to predict clinical responses to somatostatin analogs in acromegaly. Case Report We report the case of a 7-year-old boy presenting with headache, visual field defects, and accelerated growth following failure to thrive. The laboratory results showed high insulin-like growth factor I (IGF-I) (standardised deviation scores ( +3.49) and prolactin levels (0.5 nmol/L), and magnetic resonance imaging identified a pituitary macroadenoma. Tumoral/hormonal control could not be achieved despite 3 neurosurgical procedures, each time with apparent total resection or with lanreotide or pasireotide. IGF-I levels decreased with the GH receptor antagonist pegvisomant. The loss of somatostatin receptor 5 was observed between the second and third tumor resection. In vitro, no effect on tumoral GH release by pasireotide (with/without cabergoline) was observed. Genetic analysis revealed a novel germline AIP mutation: p.Tyr202∗ (pathogenic; class 4). Discussion In vitro response of tumor tissue to somatostatin may better predict tumoral in vivo responses of somatostatin analogs than somatostatin receptor immunohistochemistry. Conclusion We identified a novel pathologic AIP mutation that was associated with incipient acrogigantism in an extremely young patient who had a complicated course of disease. Growth acceleration can be masked due to failure to thrive. Tumoral growth hormone release in vivo may be predicted with in vitro exposure to somatostatin receptor analogs, as it cannot be assumed that all AIP-mutated somatotropinomas respond well to pasireotide.


Introduction
Pituitary adenomas have a prevalence of 1 clinically-relevant case per 1000 adults. 1 Most pituitary adenomas are sporadic, but 5% have a familial background, the most common being familial isolated pituitary adenomas. 1,2 In familial isolated pituitary adenomas, 15% to 30% of cases are associated with pathologic germline variants in the aryl hydrocarbon receptoreinteracting protein (AIP) gene, a tumor suppressor gene located on chromosome 11q13. 2e6 Germline AIP mutations are particularly associated with growth hormone-(GH) or mixed GH-prolactinesecreting pituitary adenomas. 3e6 Patients with AIP mutations are often men and have an aggressive clinical phenotype due to large invasive tumors. AIP mutations are the most frequent genetic cause of pituitary gigantism (29%). 2,5,7,8 In large case series, AIP-mutated pituitary adenomas usually present in adolescence or early adulthood. 9 Early pediatric presentations of patients with AIP mutations and GH-secreting pituitary adenomas are rarely described, and responses to medical and surgical management in this challenging population are not well-understood. Here, we report the challenges faced in the presentation, diagnosis, and management of a young boy with a novel AIP mutation that led to a recurrent and resistant GH-secreting macroadenoma.

Case Description
A 7-year-old boy was hospitalized for the evaluation of multiple progressive complaints over the previous 2 years, including frontal headache, fatigue, tics, leg pain, nocturnal sweating, constipation, and poor food intake. He had a normal birthweight/height following an unremarkable pregnancy, and his family history was normal. His growth curve showed normal growth until the age of 3 years, followed by a marked decrease to about À2 standardised deviation scores (SDS) at the age of 6 years (Fig. 1). Thereafter, his growth increased rapidly compared with the Dutch national standards. His parents were of modest stature (father, 170 cm and mother, 164 cm) by the current Dutch median height standards (men, 182.9 cm and women, 169.3 cm). A sellar tumor with an enlarged sella turcica was discovered (Fig. 2). Laboratory analysis showed an insulin-like growth factor I (IGF-I) level of 56.5 nmol/L (normal range [NR], 8.3-38.2; SDS, þ3.49), prolactin level of 0.5 Fig. 1. Growth chart of the patient with incipient gigantism. The initial normal growth of the patient declined from 3 to 6 years of age, but then deflected markedly upward. The patient was diagnosed at the age of 7 years. The blue arrow corresponds with LAN treatment, the purple arrow corresponds with PAS treatment, and the red arrow corresponds with PEGV surgery. One month after switching to pasireotide, the first transsphenoidal resection was performed. Two months thereafter, pegvisomant was started. After 1 month of pegvisomant, the tumor volume increased; pegvisomant was stopped, and a second resection followed. Six months thereafter, the third surgery was performed. LAN ¼ lanreotide; PAS ¼ pasireotide; PEGV surgery ¼ pegvisomant and surgery. show an elevated random GH level (48.6 mg/L). After 1 month of pegvisomant, severe headaches returned, and bitemporal hemianopsia reoccurred due to an increase in tumor volume (Fig. 2). Pegvisomant was stopped, and a second transsphenoidal resection followed (Fig. 2). The histopathologic report revealed a pituitary adenoma staining positive for GH and negative for prolactin (Fig. 3). One month after the second transsphenoidal surgery, his IGF-I level declined to 29.3 nmol/L (0.4 SDS), GH level was 2.7 mg/L, and prolactin level declined from 0.60 to 0.38 nmol/L (NR, 0.1-0.5 nmol/L). Five months after surgery, the headaches returned, and magnetic resonance imaging 1 month later showed a small remnant lateral to the right internal carotid artery (Fig. 2). IGF-I increased again to þ2.9 SDS. A third transsphenoidal surgery was performed, leading to the normalization of GH and IGF-I levels. Thirteen months after his last surgery, he received stereotactic radiotherapy (54 Gy), and 4 months after radiotherapy, his last IGF-I was À0.8 SDS.
Due to the presentation with a macroadenoma at a young age, germline genetic testing for sequence variants and deletions in AIP and MEN1 genes was performed. A novel heterozygotic truncating variant in the AIP gene was discovered (c.606C>G; p.Tyr202*; GnomAD database mean allele frequency (0), which was accompanied by a second missense variant (c.695C>T: p.Pro232Leu; mean allele frequency, 0.00002502), both of which were paternally inherited. Screening by magnetic resonance imaging and hormone evaluation of his 37-year-old father was negative.
Histopathologic analysis revealed a loss of SSTR5 expression between the second and third operations (Fig. 3). In vitro characterization of the second surgical sample showed no statistically significant inhibition of GH secretion to incubation with pasireotide (10 nM) or coincubation with pasireotide and cabergoline (both 10 nM; Fig. 4). Other compounds could not be tested due to the limited amount of available tissue. These interesting findings should be confirmed in a wider series of tumors from patients with AIP mutations and in appropriate wild-type acromegaly controls.
Given the lack of tumor size control with firstand secondgeneration somatostatin analogs (SSAs) and the unresectable remnant that required radiotherapy at the age of 10 years, the patient will require intensive (endocrinological) follow-up, although no pituitary deficiencies have occurred to date. If needed, excessive GH can be controlled by pegvisomant, albeit with high vigilance for tumor regrowth.

Discussion
This case report describes a complicated somatotropinoma leading to accelerated longitudinal growth that was masked by an unexpected initial period of failure to thrive, which likely occurred due to poor feeding because of nausea. The disease was diagnosed at the very young age of 7 years and was found to be due to a previously undescribed AIP mutation that was inherited from his unaffected father. Decreased clinical SSA sensitivity may be related to the evolving tumor biology between surgeries, particularly the loss of tumoral somatostatin receptor (SSTR) 5 expression, whereas in vitro, there was no tumoral response of GH to pasireotide and cabergoline.
Somatotropinomas are primarily treated with (transsphenoidal) neurosurgery, SSAs, dopamine agonists, or GH receptor antagonists. 11 Overall, in acromegaly, long-acting SSAs can achieve biochemical normalization of GH and IGF-I in 50% to 60% and often lead to modest tumor shrinkage. 11e14 Patients with AIP mutations have, however, significantly less tumor shrinkage and lower hormonal responses to first-generation SSAs. 7 SSAs act via SSTRs1 to 5. 6 The first-generation SSAs octreotide and lanreotide have the highest affinity for SSTR2 and have a low affinity to SSTR3 and SSTR5, whereas the second-generation SSA pasireotide has the highest affinity for SSTR5, followed by SSTR2, SSTR3, and SSTR1. 6,15 As reported previously, pasireotide resistance is possibly more related to SSTR2 expression than to SSTR5 in the general acromegaly population. 16,17 SSA resistance may occur if the tumor is lacking SSTR2. 6 Daly et al 18 recently reported 2 AIPmutated acromegaly patients with resistance to first-generation SSA, in whom pasireotide treatment led to marked tumor shrinkage and persistent hormonal control. In 1 case, very low-toabsent SSTR2 levels were seen, and the efficacy of pasireotide must have been through other SSTRs like SSTR5. 18 Due to this, we initially expected that our patient would respond better to pasireotide despite resistance to first-generation SSAs, but this was not the case. 18 The resistance to pasireotide probably relates in part to the low SSTR5 expression, since SSTR2 expression remained present. Nevertheless, signaling via SSTR2 may be affected while leaving the receptor expression unaffected. Possible factors in this phenomenon include ZAC1 and miR-34a, both of which influence SSTR2 signaling. 11,19 In these cases, it may be preferable to test the in vitro response of the tumor tissue assessed by decreases in GH secretion (17). In the study by Coopmans et al 20 that included 45 acromegaly patients who were previously treated with first-generation SSAs combined with pegvisomant, SSTR2 immunoreactivity scores were found to be related to significant tumor shrinkage in patients treated with pasireotide, which was not the case for SSTR5. Muhammad et al 16 found in the same cohort that IGF-1 lowering effects of pasireotide correlated with SSTR2 instead of SSTR5. However, the timing of the change in responsiveness and change in SSTR5 expression occurred simultaneously in the current case. In the study by Iacovazzo et al 21 that included 39 patients with somatotropinomas, SSTR5 expression predicted responsiveness to pasireotide.
Our case exemplifies the many challenges that can be faced in the recognition of acromegaly, especially when occurring at an extremely young age. Acrogigantism can occur with increased growth velocity in young patients, even without extremely elevated height compared to age-/sex-matched references. An appreciation of the totality of the abnormal growth characteristics is important when assessing children with aberrant growth. In this case, a novel AIP mutation, p.Tyr202*, was found. The unresponsiveness of the tumor to pasireotide may be better assessed by in vitro responsiveness as opposed to somatostatin receptor evaluation. Future  Growth hormone secretion by primary cultured adenoma cells of the patient did not respond to incubation with pasireotide (10 nM) or pasireotide (10 nM) plus cabergoline (10 nM); there was no statistically significant change in the growth hormone secretion after a 72-hour incubation with the drugs. The cells were cultured as a monolayer in a 250-mL medium in a 48-well culture plate. The tumor cell isolation and culture conditions were as described by Hofland et al. 17 The medium growth hormone concentrations are expressed in mg/L and are mean ± SD (n ¼ 3 wells per group). Data were analyzed by a 1-way analysis of variance, and multiple comparisons between groups were assessed with the Newman-Keuls test using GraphPad Prism. hGH ¼ human growth hormone; PAS ¼ pasireotide.
studies are necessary to test this hypothesis in cohorts with more patients and with a control group.

Conclusion
This informative case of incipient gigantism in a 7-year-old child with a novel AIP mutation, p.Tyr202*, was associated with a highly treatment-resistant somatotropinoma. Although previous literature suggests a favorable response to pasireotide in some patients with AIP mutations and acromegaly, pasireotide had only limited effect in our patient, possibly related to decreasing SSTR5 expression of the tumor. 18 In vitro GH suppression in the cultured tumor tissue may predict in vivo treatment response better than SSTR assessment. Genetic testing of the AIP gene should be advocated in all patients with GH-secreting pituitary adenomas occurring in childhood and/or (incipient) pituitary gigantism. 22