TGFBR3L is associated with gonadotropin production in non-functioning gonadotroph pituitary neuroendocrine tumours.

PURPOSE: Transforming growth factor-beta receptor 3-like (TGFBR3L) is a pituitary enriched membrane protein selectively detected in gonadotroph cells. TGFBR3L is named after transforming growth factor-beta receptor 3 (TGFBR3), an inhibin A co-receptor in mice, due to sequence identity to the C-terminal region. We aimed to characterize TGFBR3L detection in a well-characterized, prospectively collected cohort of non-functioning pituitary neuroendocrine tumours (NF-PitNETs) and correlate it to clinical data. METHODS: 144 patients operated for clinically NF-PitNETs were included. Clinical, radiological and biochemical data were recorded. Immunohistochemical (IHC) staining for FSHß and LHß was scored using the immunoreactive score (IRS), TGFBR3L and TGFBR3 were scored by the percentage of positive stained cells. RESULTS: TGFBR3L staining was selectively present in 52% of gonadotroph tumours. TGFBR3L was associated to IRS of LHß (median 2 [IQR 0-3] in TGFBR3L negative and median 6 [IQR 3-9] in TGFBR3L positive tumours, p < 0.001), but not to the IRS of FSHß (p = 0.32). The presence of TGFBR3L was negatively associated with plasma gonadotropin concentrations in males (P-FSH median 5.5 IU/L [IQR 2.9-9.6] and median 3.0 [IQR 1.8-5.6] in TGFBR3L negative and positive tumours respectively, p = 0.008) and P-LH (median 2.8 IU/L [IQR 1.9-3.7] and median 1.8 [IQR 1.1-3.0] in TGFBR3L negative and positive tumours respectively, p = 0.03). TGFBR3 stained positive in 22% (n = 25) of gonadotroph tumours with no correlation to TGFBR3L. CONCLUSION: TGFBR3L was selectively detected in half (52%) of gonadotroph NF-PitNETs. The association to LHß staining and plasma gonadotropins suggests that TGFBR3L may be involved in hormone production in gonadotroph NF-PitNETs.

A high-throughput analysis of the IDH1(R132H) protein expression in pituitary adenomas.

PURPOSE: Inactivating mutations of isocitrate dehydrogenase (IDH) 1 and 2, mitochondrial enzymes participating in the Krebs tricarboxylic acid cycle play a role in the tumorigenesis of gliomas and also less frequently in acute myeloid leukemia and other malignancies. Inhibitors of mutant IDH1 and IDH2 may potentially be effective in the treatment of the IDH mutation driven tumors. Mutations in the succinate dehydrogenase, the other enzyme complex participating in the Krebs cycle and electron transfer of oxidative phosphorylation occur in the paragangliomas, gastrointestinal stromal tumors, and occasionally in the pituitary adenomas. We aimed to determine whether the IDH1(R132H) mutation, the most frequent IDH mutation in human malignancies, occurs in pituitary adenomas. METHODS: We performed immunohistochemical analysis by using a monoclonal anti-IDH1(R132H) antibody on the tissue microarrays containing specimens from the pituitary adenomas of different hormonal types from 246 patients. In positive samples, the status of the IDH1 gene was further examined by molecular genetic analyses. RESULTS: In all but one patient, there was no expression of mutated IDH1(R132H) protein in the tumor cells by immunohistochemistry. Only one patient with a recurring clinically non-functioning gonadotroph adenoma demonstrated IDH1(R132H)-immunostaining in both the primary tumor and the recurrence. However, no mutation in the IDH1 gene was detected using different molecular genetic analyses. CONCLUSION: IDH1(R132H) mutation occurs only exceptionally in pituitary adenomas and does not play a role in their pathogenesis. Patients with pituitary adenomas do not seem to be candidates for treatment with the inhibitors of mutant IDH1.

Are volume measurements of non-functioning pituitary adenomas reliable?

PURPOSE: Precise radiological assessment of tumour volume is important in the follow-up of non-functioning pituitary adenomas (NFPAs). We compared the reliability of two methods for tumour volume measurements in the pre- and postoperative setting. METHODS: We assessed the volume of 22 NFPAs at magnetic resonance imaging (MRI) scans before surgery and the first and third postoperative MRI obtained after submission from hospital. Volumetric assessments were performed both by summation of slices (SOS) and by diameter measures. All volumes were calculated independently by two readers. RESULTS: The preoperative intra- and inter-rater reliability was good for both the SOS and the diameter method (intraclass correlation coefficient (ICC) 0.996 and 0.990, and ICC: 0.982 and 0.967, respectively). The first postoperative investigation showed poorer intra- and inter-rater reliability for both methods (ICC: 0.872 and 0.791 and ICC: 0.792 and 0.810, respectively). The third postoperative MRI showed good intra-rater reliability (ICC: 0.961 and 0.962, respectively), but poorer inter-rater reliability for both methods (ICC: 0.759 and 0.703, respectively). Volume assessment by SOS presented overall slightly higher reliability than the diametric method. Overall, the reliability between the two methods was good when measured by the same reader (ICC: 0.988, 0.945 and 0.962, for the preoperative, first and third postoperative MRI, respectively). CONCLUSION: The preoperative intra- and inter-rater reliabilities were satisfactory for both the SOS and diametric method. Postoperative MRI scans showed poorer reliability, suggesting that measurements at these time points should be interpreted with care. For each MRI scan, reliability between methods was satisfactory when investigated by the same reader.

Hip Structure Analyses in Acromegaly: Decrease of Cortical Bone Thickness After Treatment: A Longitudinal Cohort Study.

Long-standing growth hormone (GH) excess causes the skeletal clinical signs of acromegaly with typical changes in bone geometry, including increased cortical bone thickness (CBT). However, a high prevalence and incidence of vertebral fractures has been reported. The aim of this study was to assess the course of cortical bone dimensions in the hip by comparing patients with acromegaly and clinically nonfunctioning pituitary adenomas (NFPAs) at baseline and 1 year after pituitary surgery (1-year PO) in a longitudinal cohort study. DXA was performed in patients with acromegaly (n = 56) and NFPA (n = 47). CBT in the femoral neck (CBTneck), calcar (CBTcalcar), and shaft (CBTshaft) were determined by hip structural analysis (HSA). CBT at baseline and the change to 1-year PO were compared. Test results were adjusted for differences in gender distribution, age, and gonadal status. Cortical thickness analyses showed higher values [mm] at baseline in patients with acromegaly compared with NFPA: CBTneck median [25th; 75th] 6.2 [4.7; 8.0] versus 5.1 [4.1; 6.4] (p = 0.006), CBTcalcar 4.8 [4.2, 5.7] versus 4.0 [3.2, 4.5] (p < 0.001), CBTshaft 6.2 [5.1, 7.2] versus 5.2 [4.6, 6.0], (p = 0.003). In acromegaly, GH was correlated with CBTneck (r = 0.31, p = 0.020), whereas IGF-1 was correlated with CBTcalcar (r = 0.39, p = 0.003) at baseline. In acromegaly, CBTneck decreased by 11.2%, p = 0.002 during follow-up. Finally, the decrease in CBTneck and CBTcalcar in acromegaly was significant compared with NFPA (p = 0.023 and p = 0.017, respectively). Previous observations of increased CBT in acromegaly were confirmed with DXA-derived HSA in a large, well-defined cohort. The decline in CBT in acromegaly could contribute to the increased fracture risk in acromegaly despite increased bone dimensions and disease control. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

The role of E and N-cadherin in the postoperative course of gonadotroph pituitary tumours.

PURPOSE: Gonadotroph tumours are the most abundant of the clinically silent pituitary tumours. There is a lack of reliable prognostic markers predicting their clinical course. Our aim was to determine the level of E-cadherin and N-cadherin in a cohort of clinically silent gonadotroph pituitary tumours, and compare them to the rate of reintervention. METHODS: Tumour tissue from primary surgery was retrospectively investigated and compared with clinical data. Immunohistochemical (N = 105) and real time-qPCR (N = 85) analyses for the levels of N-cadherin and the extra- and intracellular domains of E-cadherin were performed. The immunoreactive scores (IRS) and mRNA relative quantity were compared to the rate of reintervention. RESULTS: The tumours presented a high IRS for N-cadherin (Median 12 (IQR 12-12)) and almost no immunoreactivity for the extracellular domain of E-cadherin (Median 0 (IQR 0-0)). The membranous staining for the intracellular domain of E-cadherin varied (Median 6 (IQR 4-6). Reduced membranous expression of the intracellular domain of E-cadherin was associated with nuclear presence of the same domain. Nuclear staining for the intracellular domain of E-cadherin was associated with a lower rate of reintervention (p = 0.01). CONCLUSION: We found that silent gonadotroph tumours presented high IRS for N-cadherin and low IRS for the extracellular domain of E-cadherin. A substantial proportion of the tumours presented nuclear staining for the intracellular domain of E-cadherin, accompanied by a reduced membranous expression of the intracellular domain of E-cadherin. Absence of nuclear staining for the intracellular domain of E-cadherin served as an independent predictor of reintervention.

Selection and validation of reliable reference genes for RT-qPCR analysis in a large cohort of pituitary adenomas.

BACKGROUND: Real-time reverse transcription quantitative PCR (RT-qPCR) has become the method of choice for quantification of gene expression changes. The most important limitations of RT-qPCR are inappropriate data normalization and inconsistent data analyses. Pituitary adenomas are common tumours, and the appropriate interpretation of increasingly published data within this field is prevented by the lack of a proper selection and validation of stably expressed reference genes. AIM: To find and validate the optimal reference gene or gene combination for reliable RT-qPCR gene expression in both non-functioning (NFPA) and hormone secreting (GH and ACTH) pituitary adenomas. MATERIAL AND METHODS: Thirty commonly used reference genes (PCR array reference gene panel, BioRad, Hercules, CA) were quantified by RT-qPCR in 24 pituitary adenomas (12 NFPA, 8 GH and 4 ACTH). The data was analysed using three programs: geNorm (Qbase+), Normfinder and BestKeeper having different algorithms to identify the most stable reference gene or combination of reference genes. Three reference genes ALAS1, PSMC4 and GAPDH, were selected for further validation in a larger cohort of 223 adenomas (141 NFPA, 63 GH and 19 ACTH). RESULTS: In all adenomas, ALAS1 and PSMC4 were the most stable reference genes as estimated by geNorm and Normfinder, whereas Bestkeeper ranked RPLP0 and ACTB as the two most stable out of 10 carefully selected genes. The best gene combination was PSMC4 and ALAS1 (geNorm) or PSMC4 and GAPDH (Normfinder). The validation experiment (geNorm) showed that the most stable gene combinations were ALAS1 and GAPDH in NFPA, and PSMC4 and GAPDH in hormone secreting adenomas. CONCLUSIONS: Several of the reference genes expressed good stability yielding several candidate genes. PSMC4 and ALAS1 were overall the most stably expressed genes in pituitary adenoma merely differing in ranking order. PSMC4 and ALAS1 have so far not been reported as reference genes in pituitary adenomas. The various reference gene algorithms showed a mixed selection of top ranked genes, thus suggesting a need for an individualised and rational choice of reference genes.