Patients
We retrospectively analyzed surgical specimens of PA obtained at Froedtert and Medical College of Wisconsin. Patients provided research consent preoperatively (institutional review board–protocol PRO00039940). Samples were selected based on the availability of a sufficient number of banked tumors to create a representative panel of various PA subtypes (Table 1, Table S1). The histological types of PA were defined based on hormone IHC and preoperative clinical and biochemical presentation. Preoperative clinical diagnostic and pathological data for each participant is presented in Table S1.
Table 1: Summary of patient characteristics.
Total number of patients
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60
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Age (mean ± SD) (years)
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51.8 ± 18.0
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Sex
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Male (N, %)
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26 (43.3%)
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Female (N, %)
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34 (56.7%)
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Clinical classification (N, %)
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NFPA
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31 (51.7%)
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GH-secreting (Acromegaly)
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7 (11.7%)
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PRL-secreting (Hyperthyrotropinemia)
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2 (3.3%)
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GH- and PRL-secreting (Acromegaly + Hyperprolactinemia)
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4 (6.7%)
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TSH-secreting
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1 (1.7%)
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ACTH secreting (Cushing’s Disease)
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15 (23.3%)
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Maximal tumor diameter (mean ± SD) (mm)
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19.72 ± 12.0
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NFPA
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21.9 ± 8.49
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Functioning
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17.34 ± 15.11
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Cavernous Sinus invasion (N, %)
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Knosp 0-2
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45 (75%)
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Knosp 3-4
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15 (25%)
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Post-operative follow-up
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Patients with sufficient follow-up (N, %)
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46 (72%)
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Average follow up length (Average, month)
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39.5
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Range (months)
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6 to 62
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Clinical course
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Recurrence
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9 (15.0%)
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Tumor (NFPAs)
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6 (10.0%)
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Biochemistcal (Functional PAs)
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3 (5%)
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The following tumor types were analyzed: 1) nonfunctioning (NFPA, i.e. no hypersecretory syndrome detected preoperatively) (n=31) and 2) functioning (n=29) as follows: a) lactotroph–causing hyperprolactinemia (n=2), b) somatotroph–causing acromegaly (n=7); c) somatotroph and lactotroph - causing acromegaly and hyperprolactinemia (n=4); d) corticotroph–causing Cushing disease (CD) (n=15), and e) thyrotroph–causing central hyperthyroidism) (n=1). Prolactinoma was defined according to prolactin levels and clinical manifestations [16]. Acromegaly was defined as high IGF-1 associated with suggestive clinical manifestations and/or lack of suppression of GH levels below 0.4 ng/mL during oral glucose challenge (75 g). CD was defined by signs and symptoms of cortisol excess and supportive screening and localization studies [17]. In cases of equivocal imaging, inferior petrosal sinus sampling was performed. Central hyperthyroidism was defined as elevated free T4 with inappropriate TSH level after excluding other causes of abnormal thyroid tests.
NFPAs were characterized as 1) Silent gonadotrophs based on the presence of SF1, LH, or FSH (n=20); 2) Silent corticotrophs based on the presence of TPIT and/or ACTH (n=2); 3) Silent thyrotrophs based on the presence of PIT1 and GATA2 and/or TSH (n=2). 4) Pluricellular adenomas containing more than one cell type (n=6). Included in pluricellular adenomas were previously described silent gonadotrophs, corticotrophs, and thyrotrophs, as well as silent somatotrophs based on immunostaining or qPCR detection of PIT1 and GH and silent lactotrophs based on immunostaining or PIT1 and/or PRL.
Pituitary Adenoma and Normal Pituitary Tissue Collection
Adult patients undergoing surgery for their PA at the Medical College of Wisconsin between 2008-2022 were included in the study. Informed consent for the collection, processing, banking and study of their tumors was obtained for each patient under an institutional review board protocol (PRO00039940). PAs were frozen and stored promptly following the resection.
Post-mortem pituitary gland collection was performed under an institutional review board–approved protocol (PRO00039940). The pituitary tissue collected post-mortem constituted either control (normal) pituitary tissue or incidental pituitary adenoma discovered at the time of autopsy.
Standard Clinical Histopathological Analysis
PA tumor tissue was analyzed based on a standard protocol at a CLIA-approved pathology laboratory. Hormones (GH, ACTH, FSH, LH, TSH, PRL) and/or transcription factor (SF1) staining was performed by IHC. Positive tumor hormone staining in patients without clinical and biochemical signs of preoperative hormone excess defined the cellular origin of nonfunctioning tumors. MIB was also stained in some tumors.
Clinical outcomes
Pre- and postoperative clinical information was retrospectively analyzed. Short-term outcomes included immediate postoperative biochemical remission for functioning adenomas and tumor residual based on the 3-month postoperative magnetic resonance imaging (MRI). Long-term outcomes included continued hormone status surveillance (persistent remission vs recurrence), and need for adjunctive management including reoperation, medications and radiation. Tumor progression was defined as either tumor recurrence following gross total resection or residual tumor growth following subtotal resection. Length of follow-up was determined by the time between index surgery and most recent imaging for NFPA and either most recent imaging or hormone measurement for functioning PA. Sufficient follow-up was defined as 6 months for the purposes of this study.
Imaging
Preoperative MRI was available for all patients. Tumor characteristics analyzed included size and degree of cavernous sinus invasion. Size was defined by maximal tumor diameter, i.e. the longest tumor axis on coronal or sagittal T1-weighted postcontrast images. Cavernous sinus involvement was graded utilizing the Knosp classification system [18] after review of coronally oriented postcontrast T1-weighted MRI, which was confirmed with a coronal T2-weighted sequence, if available. Knosp grade 0, 1, and 2 tumors which do not extend past the medial and mid and lateral intercarotid lines were considered not invasive into the cavernous sinus. Knosp 3A and 3B tumors extending past the lateral intercarotid line, and Knosp 4 PAs encasing the carotid artery were considered invasive of the cavernous sinus. For this study, all images were reviewed by a skull-base neurosurgeon.
RNA extraction
mRNA was isolated with the PureLink RNA Mini Kit (#12183018A, Thermo Fisher Scientific) following the kit instructions. RNA concentrations were measured using an LVis microplate (BMG Labtech).
cDNA preparation and qPCR
cDNA was then synthesized with SuperScript™ IV VILO™ Master Mix with ezDNase (#11766050, Thermofisher Scientific) according to the manufacturer's instructions. The resulting cDNA was diluted sequentially to a 1:250 dilution and used as templates for qPCR. qPCR reaction was then performed using PowerSYBR qPCR Master Mix (Thermo Fisher Scientific). Specific primers for each reaction are as follows: ACTIN-F: 5-GAT TCC TAT GTG GGC GAC GA-3; ACTIN-R: 5-AGG TCT CAA ACA TGA TCT GGG T-3; TPIT-F: 5-GCA AAG TGA AGC TGA CCA AC-3; TPIT-R: 5-GCA CTT CCA ACA CGC ACT AT-3; OGT-F: 5-GGT TTG AAG CCT GTA ACT GCT G-3; OGT-R: 5-TTT CGT TGG TTC TGT GCT GTC-3. qPCR was performed on a QuantStudio 3 instrument (Applied Biosystems) with the recommended settings. The data were collected and processed using DataConnect (Thermo Fisher Scientific), and 2-DDCq were calculated and plotted using Prism 9 (GraphPad Software).
RNA sequencing
Total RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina following manufacturer’s instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). Samples were processed on illumna HiSeq platform by GENEWIZ (Azenta Life Science).
Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. The STAR aligner is a splice aligner that detects splice junctions and incorporates them to help align the entire read sequences. BAM files were generated as a result of this step. Unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the various groups were performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 2 were called differentially expressed genes (DEGs) for each comparison.
Immunohistochemistry
Paraffin-embedding, sectioning, and H&E staining were performed by the children’s histology core at the Medical College of Wisconsin. Slides were first deparaffinized by successive incubation in xylene, 95% ethanol, 75% ethanol, and DI water, twice each, for 5 minutes. Antigen retrieval was done by boiling slides in Sodium Citrate (10 mM pH 6.0) for 15 minutes. After cooling, slides were run under DI water for 10 minutes. A hydrophobic barrier was drawn around the specimens with a PAP pen. The specimens were treated with 3% hydrogen peroxide for 10 minutes and rinsed twice with TBS-Tween20 (0.1%) for 2 minutes. They were then blocked with normal goat serum in PBS for 30 minutes. Specimens were incubated overnight at 4°C with Anti-OGT and Anti-OGA primary antibodies, which were diluted in blocking buffer. Slides were rinsed 3 times with PBS between each of the following steps: First, slides were incubated with a secondary biotinylated antibody in TBS-Tween20 for 10 minutes, then with Streptavidin-HRP for 10 minutes, a chromogen reagent for 10 minutes, and finally with Hematoxylin for 1 minute. Slides were then dehydrated by successive incubation in 50% ethanol, 100% ethanol, and xylene, twice each, for 2 minutes. A Permount mounting media was applied before a cover slip. Slides were dried overnight at 37°C before imaging on the EVOS M5000 microscope (Thermo Fisher Scientific). OGT antibody (Abcam - ab177941) and OGA antibody (Abcam - ab124807) were used at 1:1000 and 1:100 dilution, respectively.
Graphics and visualization
Heatmaps were done using pheatmap on R. Networks were generated using the Cytoscape app and STRING-based interaction analysis.
Gene Ontology (GO) Enrichment analysis
Publication enrichments were performed using STRING. The ClueGO app was used to create the GO networks.
Data availability
RNA-sequencing raw (*R1.fastq and *R2.fastq) and processed (*.bam and counts.txt) files were deposited in Gene Expression Omnibus (GEO) repository under GSE201439.