Objective: The most common type of pituitary diseases are pituitary adenomas (PA) of different origin. Although non-metastasizing, they still cause increased mortality and morbidity. Clinically significant PAs affect around 0.1% of population during lifetime. Circulating cell free DNA (ccfDNA) is approximately 165 bp long DNA fragments which are released in bloodstream upon cell death. ccfDNA has been used to investigate presence and properties of various cancers. Data on ccfDNA detection of adenoma of any type and localization is sparse in current literature. The primary objective of this study was to explore usability of semiconductor sequencing and CAST-PCR for detection of ccfDNA in PA using two different molecular methods.
Design: Tissue samples from adenoma of different origin of ten PA patients were screened for GNAS R201C mutation. The three positive samples were used in CAST-PCR to detect mutation in the ccfDNA. Five of the ten PA patients had exomes of germline DNA and adenoma tissue somatic DNA sequenced. Somatic mutations discovered in the exome of adenoma were also used in detection of ccfDNA.
Methods: Blood samples were collected from patients before resection of PA. Plasma, obtained by double centrifugation, were frozen within 2h after sampling. After resection, tissue samples were stored in RNAlaterSolution. ccfDNA, germline DNA and somatic DNA was extracted using QIAamp Circulating Nucleic Acid Kit, chloroform-phenol method and AllPrep DNA/RNA Mini Kit, respectively. Presence of GNAS R201C in somatic DNA was confirmed using Sanger sequencing. CAST-PCR was performed on somatic DNA and ccfDNA from positive samples, using TaqMan® Mutation Detection Assay GNASc.601C>T/ and real-time PCR. NGS Libraries were prepared with Ion Plus Fragment Library Kit. Sequences were aligned to GRCh37 using BWA. Each patient provided informed consent. Study has been approved by Central Medical Ethics Committee of Latvia (No. 22.03.07/A7, 01.29.1/28).
Results: Sixteen amplicons containing somatic mutation from five PA patients were sequenced (average coverage 6191X). Five mutations from two patients were found in approximately 50% of sequencing reads. Alternate alleles of somatic mutations located in MPRIP and RYR1 genes were detected at 3.7% and 2.3% levels, respectively. These mutations were not detected in germline DNA. Nine mutations were not detected in ccfDNA above level of technological error. CAST-PCR of GNAS R201C in plasma ccfDNA yielded negative results.
Conclusions: For the first time we demonstrate possibility to detect somatic mutations found in ccfDNA from PA patients. Whether source of mutation containing ccfDNA is PA should be studied further.
18 - 21 May 2019
European Society of Endocrinology