Transcriptome of GH-producing pituitary neuroendocrine tumours and models are significantly affected by somatostatin analogues.

Pituitary neuroendocrine tumours (PitNETs) are neoplasms of the pituitary that overproduce hormones or cause unspecific symptoms due to mass effect. Growth hormone overproducing GH-producing PitNETs cause acromegaly leading to connective tissue, metabolic or oncologic disorders. The medical treatment of acromegaly is somatostatin analogues (SSA) in specific cases combined with dopamine agonists (DA), but almost half of patients display partial or full SSA resistance and potential causes of this are unknown. In this study we investigated transcriptomic landscape of GH-producing PitNETs on several levels and functional models-tumour tissue of patients with and without SSA preoperative treatment, tumour derived pituispheres and GH3 cell line incubated with SSA to study effect of medication on gene expression. MGI sequencing platform was used to sequence total RNA from PitNET tissue, pituispheres, mesenchymal stromal stem-like cells (MSC), and GH3 cell cultures, and data were analysed with Salmon-DeSeq2 pipeline. We observed that the GH-producing PitNETs have distinct changes in growth hormone related pathways related to its functional status alongside inner cell signalling, ion transport, cell adhesion and extracellular matrix characteristic patterns. In pituispheres model, treatment regimens (octreotide and cabergoline) affect specific cell proliferation (MKI67) and core functionality pathways (RYR2, COL8A2, HLA-G, ARFGAP1, TGFBR2). In GH3 cells we observed that medication did not have transcriptomic effects similar to preoperative treatment in PitNET tissue or pituisphere model. This study highlights the importance of correct model system selection for cell transcriptomic profiling and data interpretation that could be achieved in future by incorporating NGS methods and detailed cell omics profiling in PitNET model research.

Genome wide analysis of circulating miRNAs in growth hormone secreting pituitary neuroendocrine tumor patients' plasma.

Background: Circulating plasma miRNAs have been increasingly studied in the field of pituitary neuroendocrine tumor (PitNET) research. Our aim was to discover circulating plasma miRNAs species associated with growth hormone (GH) secreting PitNETs versus assess how the plasma levels of discovered miRNA candidates are impacted by SSA therapy and whether there is a difference in their levels between GH secreting PitNETs versus other PitNET types and healthy individuals. Design: We compared plasma miRNA content and levels before and after surgery focusing on GH secreting PitNET patients. Selected miRNA candidates from our data and literature were then tested in a longitudinal manner in somatostatin analogues (SSA) treatment group. Additionally, we validated selected targets in an independent GH secreting PitNET group. Methods: miRNA candidates were discovered using the whole miRNA sequencing approach and differential expression analysis. Selected miRNAs were then analyzed using real-time polymerase chain reaction (qPCR). Results: Whole miRNA sequencing discovered a total of 16 differentially expressed miRNAs (DEMs) in GH secreting PitNET patients' plasma 24 hours after surgery and 19 DEMs between GH secreting PitNET patients' plasma and non-functioning (NF) PitNET patients' plasma. Seven miRNAs were selected for further testing of which miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p showed a significant downregulation in plasma after 1 month of SSA treatment. mir-625-5p was found to be significantly downregulated in plasma of GH secreting PitNET patients vs. NF PitNET patients. miR-625-5p alongside miR-130b-3p were also found to be downregulated in GH PitNETs compared to healthy individuals. Conclusions: Our study suggests that expression of plasma miRNAs miR-625-5p, miR-503-5p miR-181a-2-3p and miR-130b-3p in GH secreting PitNETs is affected by SSA treatment. Additionally, miR-625-5p can distinguish GH secreting PitNETs from other PitNET types and healthy controls warranting further research on these miRNAs for treatment efficacy.

Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors.

Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0-19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs.

Case report: recurrent pituitary adenoma has increased load of somatic variants.

BACKGROUND: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. In this study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in the same patient to explain the causality mechanisms of PA recurrence. CASE PRESENTATION: The patient was a 69-year-old female with non-functional pituitary macroadenoma with extension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August 2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knosp grade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In both tumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germline DNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling were performed followed by variant filtering and manual review with IGV software. We observed an increased load of missense variants in the recurrent PA tumour when compared to the original tumour. The number of detected variants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally, targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encoded proteins. CONCLUSIONS: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression. The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused by clonal expansion of the leftover tumour tissue.

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue.

Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein-protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein-protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.

Evaluation of the Possibility to Detect Circulating Tumor DNA From Pituitary Adenoma.

Objective: Circulating free DNA (cfDNA) in general and circulating tumor DNA (ctDNA) in particular is becoming an increasingly used form of liquid biopsy biomarkers. In this study, we are investigating the ability to detect ctDNA from the plasma of pituitary adenoma (PA) patients. Design: Tumor tissue samples were obtained from planed PA resections, before which blood plasma samples were taken. Somatic variants found in PA tissue samples were evaluated in related cfDNA, isolated from plasma samples. Methods: Sanger sequencing, as well as previously obtained whole-exome sequencing data, were used to evaluate somatic variants composition in tumor tissue samples. cfDNA was isolated from the same PA patients and competitive allele-specific TaqMan PCR and amplicon-based next-generation sequencing (NGS) approach were used for targeted detection of variants found in corresponding tumor tissue samples. Results: Using NGS-based analysis, we detected five out of 17 somatic variants in 40 to 60% of total reads, three variants in 0.50-5.00% of total read count, including GNAS c.601C>T, which was detected using ultra-deep NGS (1.78 million X) in 0.77% of amplicons reads. Nine variants were not detected. We also detected We were not able to detect variant found in PA tissue in cfDNA using cast-PCR, indicating that the portion of variant-containing ctDNA in total isolated cfDNA is too small to be detected with this method. Conclusions: For the first time, we demonstrate the possibility to detect somatic variants of PA in cfDNA isolated from patients' blood plasma. Whether the source of variant detected in cfDNA is PA should be further tested.