Whole-genome Sequencing of Follicular Thyroid Carcinomas Reveal Recurrent Mutations in MicroRNA Processing Subunit DGCR8

Abstract

Background: The genomic and transcriptomic landscape of widely invasive follicular thyroid carcinomas (wiFTCs) and Hürthle cell carcinoma (HCC) are poorly characterized, and subsets of these tumors lack information on genetic driver events.

Objective: The aim of this study was to bridge this gap.

Methods: We performed whole-genome and RNA sequencing and subsequent bioinformatic analyses of 11 wiFTCs and 2 HCCs with a particularly poor prognosis, and matched normal tissue.

Results: All wiFTCs exhibited one or several mutations in established thyroid cancer genes, including TERT (n = 4), NRAS (n = 3), HRAS, KRAS, AKT, PTEN, PIK3CA, MUTYH, TSHR, and MEN1 (n = 1 each). MutSig2CV analysis revealed recurrent somatic mutations in FAM72D (n = 3, in 2 wiFTCs and in a single HCC), TP53 (n = 3, in 2 wiFTCs and a single HCC), and EIF1AX (n = 3), with DGCR8 (n = 2) as borderline significant. The DGCR8 mutations were recurrent p.E518K missense alterations, known to cause familial multinodular goiter via disruption of microRNA (miRNA) processing. Expression analyses showed reduced DGCR8 messenger RNA expression in FTCs in general, and the 2 DGCR8 mutants displayed a distinct miRNA profile compared to DGCR8 wild-types. Copy number analyses revealed recurrent gains on chromosomes 4, 6, and 10, and fusion gene analyses revealed 27 high-quality events. Both HCCs displayed hyperploidy, which was fairly unusual in the FTC cohort. Based on the transcriptome data, tumors amassed in 2 principal clusters.

Conclusion: We describe the genomic and transcriptomic landscape in wiFTCs and HCCs and identify novel recurrent mutations and copy number alterations with possible driver properties and lay the foundation for future studies.

Keywords: cancer; carcinoma; follicular; mutation; thyroid; whole-genome.

Figure 1.

Heat map of the somatic mutational landscape across the whole-genome sequenced tumor cohort. Each column represents one patient/tumor. Each row represents a mutated gene and the color code represents the mutation type. The top grid displays thyroid-related genes (top 20 COSMIC mutated genes), while the bottom grid displays top mutations called by MutSigCV2 sorted by P value. Cases 202 and 208 are Hürthle cell carcinomas; the remaining 11 cases are widely invasive follicular thyroid carcinomas (wiFTCs).

Figure 2.

The landscape of copy number alterations (CNAs) in the whole-genome sequenced cohort. Summarized gain and loss events across the genome (columns represent cytobands) for each patient/tumor (rows). The bar plot on top shows the total number of CNA across each chromosome. Note the frequent gain of regions 4p11, 6p21.32, and 10q11.21 across the cohort. Cases 202 and 208 are Hürthle cell carcinomas; the remaining 11 cases are widely invasive follicular thyroid carcinomas (wiFTCs).

Figure 3.

Gene fusion events across the tumor genome. Lines represent the location of gene fusion and colors correspond to structural variation type. 28 high-confidence events were observed. Cases 204 and 205 were found to exhibit the established PAX8-PPARγ fusion (between chromosomes 2q13 and 3p25).

Figure 4.

DGCR8 messenger RNA (mRNA) expression in an extended follicular tumor cohort. The DGCR8 mRNA expression is significantly lower in follicular thyroid carcinoma (FTC) compared to follicular thyroid adenoma (FTA). The DGCR8-mutated cases are marked with blue. These cases displayed near median expression.

Figure 5.

Transcriptome analyses of follicular thyroid carcinoma, Hürthle cell carcinoma, and normal thyroid tissue. A, Unsupervised cluster analysis of tumor and normal (N) tissue. Three main clusters are seen; the 2 normal samples clustered together with a single follicular thyroid carcinoma (FTC) (case 105), whereas the remaining tumors aggregated in 2 principal clusters. Three out of 4 samples with the TERT promoter (TERTp) mutation (102, 201, and 203) clustered together. The 2 Hürthle cell carcinomas (cases 202 and 208) appeared in the same cluster as 4 widely invasive FTCs. B, Volcano plot displaying significantly upregulated and downregulated genes. Genes labeled with red dots show a fold change greater than 1 and a P value of less than .01 One of the 2 tumor clusters (containing the majority of the TERTp-mutated cases) was defined by more pronounced expression in the significantly upregulated genes. C, Enrichment analysis of upregulated genes revealed associations with genes associated with mitochondrial transmembrane transport, carnitine shuttle, and fatty acid transmembrane transport.

Figure 6.

MicroRNA (miRNA) profiling in follicular thyroid carcinoma (FTC). A, Unsupervised clustering of the top 50 most variable miRNAs in follicular thyroid carcinoma (FTC) (n = 11). The annotation on top indicates the DGCR8 p.E518K–mutated cases, which clustered together and show a general downregulation of miRNA. Nearby cases 105 and 106 both showed low DGCR8 messenger RNA (mRNA) expression levels from the transcriptomic data. Case 201 displayed augmented upregulation compared to other cases. This case showed DGCR8 mRNA expression levels similar to that of normal thyroid tissues. B, Differential expression analysis in DGCR8 p.E518K–mutated cases (n = 2) compared to DGCR8 p.E518 wild-type cases (n = 9). Only 12 miRNAs were differentially expressed between groups, possibly because of a general downregulation of miRNA in cases 105 and 106. Fold change cutoff was set to –1.5 and 1.5, and the threshold for adjusted P value was set to .05.

Figure 7.

Tumors with gain vs no gain in the 10q11.21 cytoband were analyzed for differentially expressed genes in the same cytoband. RASSF4, TMEM72, and OR13A1 were all significantly upregulated in the tumor cohort when analyzing RNA sequencing data, suggesting that the augmented expression of one or several of these genes might be associated with recurrent 10q11.21 gains observed in the cohort.

Figure 8.

Schematic overview of microRNA (miRNA) regulators in wild-type thyrocytes and mutated follicular thyroid carcinoma (FTCs). The left aspect depicts normal miRNA processing, in which miRNAs are transcribed, forming a pri-miRNA, which is subsequently targeted by Drosha and DGCR8. This molecular complex cleaves the pri-miRNA into pre-miRNA. The pre-miRNA is transported to the nucleus, where it is processed into mature miRNA by DICER. This mature miRNA sequence interacts with the RNA-induced silencing complex (RISC) and inhibits translation of target messenger RNAs (mRNAs), thereby regulating gene expression. The right aspect displays the potential functional consequences of DROSHA, DGCR8, and DICER1 mutations in FTCs. As the mutations in theory lead to the inactivation of the corresponding proteins, a defect in miRNA processing would affect gene expression output. Image created using BioRender.com.