Abstract 2390: The polyclonal nature of multinodular goitre revealed by DICER1 sequencing
Authors: de Kock Leanne , Bah Ismaël , Wu Mona , Priest John R. , Ragoussis Jiannis , Foulkes William D. 
Published: July 2016

Abstract
Nontoxic multinodular goitre (MNG) occurs frequently in the general population and is characterised by nodular enlargement of the thyroid gland. In 2011, Rio Frio et al discovered that familial MNG and MNG occurring with Sertoli-Leydig cell tumours were associated with germ-line mutations in the microRNA-processing gene, DICER1. We recently reported the first instance of biallelic DICER1 mutations in an MNG. This prompted us to further investigate the role of DICER1 mutations in MNG pathogenesis.
Given that MNG may either be a monoclonal or polyclonal disease, we set out to investigate whether the molecular landscape was correspondingly heterogeneous. We obtained core biopsies (n = 27) from 9 MNGs. 1mm cores were excised from morphologically distinct regions, including from areas of follicular hyperplasia (FH), hyperplasia within colloid pools (HC) and/or otherwise unremarkable areas. DNA extracted from each core was processed using a custom Fluidigm Access Array followed by deep sequencing. We focused our investigation on the RNase IIIb domain of DICER1 to identify known hotspot mutations. All mutations were validated by Sanger sequencing.
We identified 6 individually distinct DICER1 RNase IIIb hotspot mutations in 14 (of 27) cores consisting of FH or HC (Table 1). These mutations affected the metal-ion binding residues at positions 1705, 1709, 1810 & 1813. Two MNGs that arose in individuals without germ-line DICER1 mutations did not carry any RNase IIIb hits. Of the remaining 7 germ-line DICER1-mutated cases, 2 carried 1 RNase IIIb mutation; 4 carried 2 different RNase IIIb mutations and one case did not carry any successfully-validated RNase IIIb mutations.
We demonstrate that spatial heterogeneity of RNase IIIb DICER1 mutations exists in MNG. We postulate that cyclic hyperplasia followed by regression over time in response to external stimuli, particularly in germ-line DICER1 mutation carriers, contributes to the polyclonal accumulation of RNase IIIb mutations in MNG. Table 1:DICER1 RNase IIIb Mutation FrequenciesCaseGerm-line DICER1 StatusMorphologyc.5113G&gt;A p.E1705Kc.5125G&gt;A p.D1709Nc.5126A&gt;G p.D1709Gc.5428G&gt;T p.D1810Yc.5429A&gt;T p.D1810Vc.5437G&gt;C p.E1813Q1PositiveHC41.7–––––FH–––––22.12PositiveFH––––––UR––––––HC––––––UR––––––HC––Sanger–––HC––Sanger–––3PositiveHC––––29.2–FH–43.7––––FH–28.8––––4PositiveHC––30.6–––UR––––––HC––33.1–––FH–––52.3––5PositiveHC––––––HC with FH––––––6PositiveFH–––––39.3FH–––––13.1FH––––––7PositiveFH–––41.8––FH28.0–––––8NegativeFH––––––HC––––––9NegativeFH––––––PTC––––––FH––––––Abbreviations: FH, follicular hyperplasia; HC, hyperplasia within colloid pools; UR, unremarkable; PTC, papillary thyroid ca. Notes: Each row corresponds to a separate core. Mutant allele frequencies are indicated in each cell (all &gt;10%); ‘‘–’’ indicates mutation not observed in core DNA; Sanger, indicates mutations previously identified in an independent PCR from bulk MNG DNA, tested by Sanger sequencing. All Fluidigm positives were confirmed by Sanger sequencing from the same DNA that was used to do the Fluidigm analysis.
Citation Format: Leanne de Kock, Ismaël Bah, Mona Wu, John R. Priest, Jiannis Ragoussis, William D. Foulkes. The polyclonal nature of multinodular goitre revealed by DICER1 sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2390.