ECE2019 Poster Presentations Adrenal and Neuroendocrine Tumours 1 (60 abstracts)
Exome analysis of pituitary adenoma tissue derived cell cultures
Vita Rovite 1 , Raitis Peculis 1 , Ilona Mandrika 1 , Ramona Petrovska 1 , Kaspars Megnis 1 , Inga Balcere 2, , Ilze Konrade 2, , Janis Stukens 4 , Austra Breiksa 4 , Jurijs Nazarovs 4 , Valdis Pirags 4, & Janis Klovins 1
1Latvian Biomedical Research and Study Centre, Riga, Latvia; 2Riga Eastern Clinical University Hospital, Riga, Latvia; 3Riga Stradins University, Riga, Latvia; 4Pauls Stradins Clinical University Hospital, Riga, Latvia; 5University of Latvia Faculty of Medicine, Riga, Latvia.
Introduction: Pituitary adenomas (PA) are tumours of the anterior pituitary. Despite the benign nature these neoplasms cause increased mortality and morbidity. Clinically relevant PAs affect around 0.1% of population during their lifetime. Currently, there is no human PA cell culture models. Tissue cultures derived from PA surgery materials depending on harvesting conditions can form free floating aggregates called pituispheres (PS) or adherent mesenchymal stromal cells (MSC). We studied genetic relationship between patients’ germline DNA, tumour tissue somatic DNA, DNA of PS and MSC obtained from culture of primary surgery material to investigate usability of these culture cells as human PA model.
Methods: Five PA patients were enrolled to national biobank – Genome Database of Latvian Population from Pauls Stradins Clinical University Hospital where transsphenoidal surgery of PA was performed for all patients. Germline DNA was isolated from white blood cells using phenol-chloroform method, tumour somatic DNS was isolated using AllPrep DNA/RNA Mini Kit (Qiagen, Netherlands). To obtain PS primary PA tissue material was harvested using EGF, FGF, and B-27 supplement methodology, but adherent MSCs were developed using DMEM/serum supplemented culturing. PS and MSC were used as DNA source in whole genome amplification (WGA) to obtain sufficient DNA for library preparation. Exomes (Illumina TruSeq_Rapid_Exome_TargetedRegions_v1.2) of germline, tumour somatic, PS and MSC were sequenced using Illumina NextSeq with 75bp paired end reads. Sequencing data were analyzed with Illumina Basespace Enrichment App (v3.0.0) aligning to human HG19 reference genome using Isaac Genome Alignment Software, variants called with Starling algorithm and variants annotated with Illumina Annotation Engine. Filtered variants were reviewed using IGV 2.3.14.
Results: Germline and somatic DNA sequencing captured median 98.4% of the target regions (range 96.4–99.0%). WGA region capture was lower with median 95.6% (range 79.6–99.2%). Variation analysis revealed low amount of somatic mutations (median 4, range 0–5) in the PAs. Somatic mutations of the primary tumour can be detected in the respective PS, but not in the respective MSC. Genetic alterations of MSCs corresponded to mutations in PA patients’ germline DNA.
Conclusions: For the first time we show that genome of PS represents genome of PA while MSC derived from the same primary surgery material does not contain PA characterizing mutations in their genome therefore most likely representing normal cells of pituitary or surrounding tissues. This indicates that PS can be used as a model to study PAs.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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