3.1 Clinical characteristics

The clinical characteristics of the patients are shown in Table 1. A total of 225 TC samples (215 PTC, 6 MTC, 3 ATC, and 1 FTC) were analyzed. Of the included patients, 82(36.44%) were male and 143(63.56%) were female, and the average age was 43.85 ± 13.25 years.

TABLE 1. The relationship between clinical characteristics and mutations of TC patients Characteristics Number of patients Number of patients with no mutation Number of patients with one mutation Number of patients with more than one mutation p value Age ≥55 52(23.11%) 8 29 15 p <.001 <55 173(76.89%) 36 128 9 Gender Male 82(36.44%) 14 56 12 p = .309 Female 143(63.56%) 30 101 12 Pathological subtype PTC 215(95.56%) 40 152 23 p = .121 MTC 6(2.67%) 3 3 0 ATC 3(1.33%) 0 2 1 FTC 1(0.44%) 1 0 0 AJCC cancer stage I–II 198(88%) 37 151 10 p <.001 III–IV 27(12%) 7 6 14 Note: Pearson's chi-squared test was used, p values less than .05 were considered statistically significant. 3.2 Gene spectrum in general

In general, 207 mutations of all types of target genes were detected in 181 samples (80.44% of 225 samples), including 200 mutations in 175 PTC samples (81.40% of 215 PTC samples), 3 mutations in 3 MTC samples (50.00% of 6 MTC samples), and 4 mutations in 3 ATC samples (100% of 3 ATC samples; Figure 1). There were 44 samples (19.56% of 225 samples) without any mutations in target genes. And 157 samples (69.78% of 225 samples) harbored mutations in single gene. A total of 22 samples (9.78% of 225 samples) simultaneously carried two gene mutations and two samples (0.89% of 225 samples) had triple different gene mutations (Figure 2(A)).

Gene mutations in subtypes of TC

(A) Proportion of patients carrying different gene mutations. (B) The number of mutations in different target genes

The majority of mutation type was point mutation (94.69%, 196/207), and the other mutation type was fusion (5.31%, 11/207).The BRAF mutation was the most common type (77.78%, 161/207), followed by TERT (8.21%, 17/207), RET (6.76%, 14/207), NRAS (1.45%, 3/207), TP53 (1.45%, 3/207), GNAS (0.97%, 2/207), HRAS (0.97%, 2/207), CTNNB1 (0.97%, 2/207), PIK3CA (0.97%, 2/207), and NTRK1 (0.48%, 1/207) (Figure 2(B)).

For PTC, BRAF mutations were predominant, followed by TERT, RET fusion, RET point mutation, NRAS, TP53, PIK3CA, GNAS, NTRK1 fusion, CTNNB1, and HRAS (Table 2). And the BRAF mutations were all typical p.V600E mutations. TERT mutations are more prevalent in advanced PTC. RET fusions were only observed among the PTC cases, including CCDC6-RET fusions (n = 8), NCOA4-RET fusion (n = 1), and TRIM27-RET fusion (n = 1). Twenty-one PTC patients (9.77%, 21/215) had two gene mutations simultaneously, including BRAF/TERT (n = 14), BRAF/RET point mutation (n = 3), BRAF/PIK3CA (n = 1), BRAF/CTNNB1 (n = 1), BRAF/TP53 (n = 1), and BRAF/GNAS (n = 1). Two PTC patients (0.93%, 2/215) harbored three gene mutations simultaneously, including NRAS/TERT/TP53 mutations and BRAF/TERT/PIK3CA mutations.

TABLE 2. Target gene mutations in four pathological subtypes of TC PTC MTC ATC FTC Total BRAF 159 2 161 TERT 17 17 RET fusion 10 10 RET point mutation 3 1 4 NRAS 3 3 TP53 2 1 3 PIK3CA 2 2 GNAS 1 1 2 HRAS 1 1 2 CTNNB1 1 1 2 NTRK1 fusion 1 1 Total 200 3 4 0 207

For MTC and ATC, the type of mutations was point mutations only. In six MTC samples, three samples were positive for mutations including RET point mutations, CTNNB1, and HRAS mutations (Table 2, Figure 1). Three ATC samples were analyzed, revealing all samples positive for mutations. And one sample carried two mutations in TP53 and GNAS, two samples were positive for BRAF mutation (Table 2). No mutation was detected in FTC case (Figure 1).

3.3 Mutation allele frequency

All mutations were heterozygous mutations present with allelic frequency that ranged from 1.12 to 50.13% of alleles (which corresponds to 2.24–100.26% of cells with a heterozygous mutation). One RET point mutation (p.G691S) showed allelic frequency of more than 50% (50.13%) which was a germline variant. The fusion mutations showed the percent of reads ranged from 2.30 to 55.54%. For samples carried more than one gene mutations, the allelic frequency of mutants were almost similar. However, for sample harbored NRAS, TERT, and TP53 mutations simultaneously, the allelic frequency of NRAS mutation was similar to TERT mutation (33.62–39.67%), a lower allele frequency (4.13%) was detected in TP53 mutation.

3.4 The relationship between clinical characteristics and mutations of TC patients

Multiple mutations in TC patients were significantly more frequent in cases of patients aged 55 and over (p <.001) and advanced AJCC cancer stage (p <.001). However, gender (p = .309) and pathological subtype (p = .121) did not show significant correlation with mutations (Table 1).