Exome analysis of pituitary adenoma tissue derived cell cultures

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 somaticDNS 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.