Widespread Chromosomal Losses and Mitochondrial DNA Alterations as Genetic Drivers in Hürthle Cell Carcinoma

Abstract

Hürthle cell carcinoma of the thyroid (HCC) is a form of thyroid cancer recalcitrant to radioiodine therapy that exhibits an accumulation of mitochondria. We performed whole-exome sequencing on a cohort of primary, recurrent, and metastatic tumors, and identified recurrent mutations in DAXX, TP53, NRAS, NF1, CDKN1A, ARHGAP35, and the TERT promoter. Parallel analysis of mtDNA revealed recurrent homoplasmic mutations in subunits of complex I of the electron transport chain. Analysis of DNA copy-number alterations uncovered widespread loss of chromosomes culminating in near-haploid chromosomal content in a large fraction of HCC, which was maintained during metastatic spread. This work uncovers a distinct molecular origin of HCC compared with other thyroid malignancies.

Keywords: Hürthle cell; chromosomal losses; complex I; haploid; loss of heterozygosity; metastasis; mitochondria; mtDNA; oncocytic; thyroid cancer.

Figure 1.. The genomic landscape of HCC.

(A) Top, mutation status for significantly mutated nuclear genes using unrestricted hypothesis testing across the cohort (black font) and restricted hypothesis testing on known cancer genes (green font) are shown. Middle, genes previously associated with thyroid cancer, in which at least one non-silent mutation that appeared ≥3 times in the COSMIC database (‘recurrent mutation’), are depicted. Bottom, mitochondrial DNA alterations, TERT promoter mutations, TERT amplifications, and fusion genes are identified. Mitochondrial alterations shown are those likely disrupting complex I, including missense mutations. The * highlights the inclusion of three leucyl-tRNA variants associated with the disease MELAS (brown boxes). The detection of a single fusion gene (PAX8-PPARγ) is depicted. The white boxes denote patients with insufficient DNA coverage for analysis. (B) Top, mean tumor ploidy is shown, based on ABSOLUTE-generated ploidy values across all tumors from each patient. Histogram at left shows the distribution of mean ploidy values colored according to thresholds shown at right. Bottom, mean number of chromosomal clonal arm-level LOH events is shown (based on ABSOLUTE and averaged across all samples from each patient). Histogram at left shows distribution of mean LOH values, colored according to thresholds shown at right. Each column represents one ‘aggregated tumor’ (the union of mutations identified in all tumors from each patient, see text). In each subpanel colored boxes indicate the presence of a genomic alteration. Percentages indicate the fraction of aggregated tumors with an alteration in a gene; histograms indicate the number of alterations in a gene across the cohort of aggregated tumors. See also Figure S1 and Tables S1–S3.

Figure 2.. Widespread loss of chromosomes in HCC.

ABSOLUTE total copy numbers of individual segments are delineated by their genomic position along the 22 chromosomes (top to bottom). Sample identifiers and classifications are shown at top. Dark grey vertical lines separate different patients, and white vertical lines separate individual tumors from the same individual. Patients are ordered according to mean ploidy of ‘aggregated tumors’, and individual tumor samples from the same patient are ordered based on date of resection. The presence of WGD is indicated. Ploidy results of FISH and Imaging Flow Cytometry (Flow) are indicated by letters for near-haploid (H), diploid (D), and complex (C) tumors. Colors indicate the loss, copy neutral LOH or gain at genomic loci. * HCC-88 LR1 analysis was confounded by low tumor purity. See also Figure S2 and Table S4.

Figure 3.. Experimental validation of the near-haploid state.

Left column, genomic plots show coverage-derived somatic copy number ratios (upper panel, normalized to two by convention), and the allelic ratio of single-nucleotide variants across each chromosome (lower panel) for three representative tumor samples with near-haploid, diploid, and complex chromosomal content. Purple color represents heterozygous SNVs with allelic ratio ~0.5, whereas red represents higher allelic ratio and blue represents lower allelic ratio. Middle column, FISH images for chromosomes 4 (PDGFRA; red), 7 (EGFR; green), and 8 (centromeric; white) are shown. White arrows indicate the probe signals for the modal cell population and scale bar corresponds to 10 μM. Right column, frequency distribution histograms from imaging flow cytometry with propidium iodide (PI) labeling of nDNA are shown. Horizontal lines demarcate the gating for the modal cell population and the modal PI intensity of the parent population is shown within each plot. The vertical dashed gray line corresponds to the modal PI intensity in the diploid tumor. Bright field and fluorescent PI images confirmed measurement of single cells and representative gated cells are shown in the top right a with 10 μM scale bar. The same tumor is represented across each row. See also Figure S3.

Figure 4.. Mitochondrial DNA mutations in HCC.

(A) Mutations in mtDNA genes (VAF≥0.3) are shown, along with selected nuclear gene mutations. TERT events (shown in black) include promoter variants and gene amplifications. Each column represents one ‘aggregated tumor’. (B) Barplot shows mean number of somatic mutations in HCC samples and the pan-cancer analysis (Ju et al., 2014). Error bars show standard error; heteroscedastic two-sided t-test. (C) Barplots show percent of HCC samples containing different subsets of somatic mutations; Fisher’s Exact Test. (D-E) Comparison of variant allele frequency between somatic mutations (D) and amino acid conservation scores for sites with somatic missense mtDNA mutations (E) in HCC and the pan-cancer analysis. Horizontal lines show the median; two-sided Wilcoxon rank sum test. (F) Location of missense mutations observed in HCC within the complex I crystal structure from Thermus thermophilus (Baradaran et al., 2013). mtDNA-encoded genes (ND1-ND6) shown in colors, mutations shown as red spheres, Fe-S clusters as brown/golden spheres. Structurally similar proteins (ND2, ND4, ND5) are shown superimposed to show mutation clustering. Panels B-E show data from one index sample per patient, rather than ‘aggregated’ tumors (panel A). ns indicates non-significant (p > 0.05). See also Figure S4 and Table S5.

Figure 5.. The near-haploid genotype is stable during metastatic progression.

ABSOLUTE-derived copy numbers for allele 1 (A1, low copy numbers) and allele 2 (A2, normal or high copy numbers) are shown for all tumors obtained from each patient. Timeline of resection for each patient shown at top. Chromosomal segments are shown horizontally by genomic position for each tumor sample, with color indicating the copy number. Blue indicates 0N, red indicates 2N, grey represents 1N, and dark red represents > 3N (the color legend at bottom applies to all panels). Tumor families depicted are representative of a stable near-haploid genotype (top) and complex genotypes with WGD (bottom).

Figure 6.. The phylogeny of HCC tumor families.

(A) Phylogenetic trees show relationships between distinct tumor samples obtained from each patient. The length of the phylogenetic branches is based on the Jaccard distance between clones taking into account clonal nDNA events (CCF≥0.9). Clonal (black) and subclonal (green) gains, losses and oncogenic mutations are annotated onto the branches on which they occurred. High VAF (>0.3) complex I mtDNA mutations and TERT promoter mutations were manually added to the trees (purple). The top black dot represents the germline (normal) genome. The initial vertical line from the normal genome to the first divergence of the lines depicts events shared by all samples; lines between nodes depict events shared among a few; purple dots depict events unique to the individual tumors that are noted at the end. Below each tree, a timeline depicts the sequence of diagnosis and tissue sampling. P, primary tumor; DM, distant metastasis; LR, locoregional recurrence; LN, lymph node. (B) Barplot shows a summary of genomic alterations observed in HCC evolution, based on14 patients from whom multiple tumors were analyzed. See also Figure S5.