Clinicopathological Features of CCDC6-RET and NCOA4-RET Fusions in Thyroid Cancer: A Single-Center Retrospective Cohort Study in a Chinese Population

Abstract

Background:

The rearranged during transfection (RET) proto-oncogene fusion is common in papillary thyroid cancer (PTC), varying across ethnic groups. However, comprehensive comparisons of RET fusion types are limited. This study aims to identify predominant RET fusions and analyze their clinicopathological characteristics in a cohort of Chinese thyroid cancer cases.

Methods:

This single-center retrospective cohort study analyzed thyroid cancer data, utilizing next-generation sequencing on formalin-fixed, paraffin-embedded tissue samples. Detailed clinicopathological data of thyroid cancer cases with RET fusions were collected.

Results:

Among 2300 thyroid cancer cases, RET fusions were exclusively found in PTC or differentiated high-grade thyroid carcinoma (DHGTC) cases (2234 cases), absent in other types (66 cases). Of the 2234 PTC or DHGTC cases, 113 (5.06%) exhibited RET fusions, including 100 primary cases. Coiled-coil domain containing 6 (CCDC6)-RET fusions predominated (78.0%, 78/100), with nuclear receptor coactivator 4 (NCOA4)-RET fusions representing 22.0% (22/100). NCOA4-RET fusions were more prevalent in patients aged 45 years and older (54.5% vs. 28.2%, p = 0.021) and DHGTC cases (p < 0.05) and associated with higher rates of lymph node metastases (90.9% vs. 67.9%, p = 0.032). CCDC6-RET fusion exhibited a higher prevalence of Hashimoto’s thyroiditis (HT) (67.9% vs. 22.7%, p < 0.001) and elevated thyroglobulin antibody levels (14.11 [1.86–174.32] IU/mL vs. 2.01 [1.14–15.41] IU/mL, p = 0.018). Moreover, CCDC6-RET fusion predominantly occurred in classical PTC (56.4%, 44/78) and infiltrative follicular PTC (17.9%, 14/78), whereas NCOA4-RET fusion was more frequent in classical PTC (36.4%, 8/22), solid PTC (27.3%, 6/22), and DHGTC (27.3%, 6/22). RET fusions with compound mutations were associated with older age (≥45 years) and bilateral thyroid involvement. Follow-up data showed a higher recurrence rate in the RET fusion group compared with the BRAF^V600E mutation group (5.0% vs. 0.0%, p = 0.018). Although the NCOA4-RET group showed a numerically higher recurrence rate compared with CCDC6-RET (9.1% vs. 3.8%), this difference was not statistically significant (p = 0.559).

Conclusions:

RET fusions are specific to PTC or DHGTC cases among Chinese thyroid cancer cases. CCDC6-RET and NCOA4-RET fusions exhibited distinct clinicopathological features, with NCOA4-RET being more aggressive.

Introduction

The rearranged during transfection (RET) proto-oncogene plays a pivotal role in thyroid cancers, characterized by the following two aberrant forms: RET mutation or fusion.^1,2 RET fusions are typically identified in 6.80% of patients with papillary thyroid cancer (PTC), with sporadic occurrences in poorly differentiated thyroid cancer (PDTC) and rare cases of anaplastic thyroid cancer (ATC).^3

–6 Compared to the BRAF^V600E mutation, RET fusions are associated with greater aggressiveness, predisposing patients to lymph node and distant metastases in thyroid cancer.7,8 In addition, RET fusions are related to early recurrence and reduced 5-year disease-free survival rates in thyroid cancer patients.⁸

Several studies have demonstrated that RET rearrangements in PTC involve diverse partner genes.^9
–11 Among these, coiled-coil domain containing 6 (CCDC6) and nuclear receptor coactivator 4 (NCOA4) are the most prevalent, accounting for 90% of cases.^12,13 Notably, CCDC6-RET and NCOA4-RET fusions exhibit distinct characteristics in PTC. Specifically, CCDC6-RET fusion is more frequently observed in classical PTC and sporadic thyroid cancer, whereas NCOA4-RET fusion is associated with solid PTC and an advanced stage of disease at diagnosis.^1,14,15

Although RET fusions have been documented in PTC patients of diverse ethnic backgrounds, reports on Chinese thyroid cancer patients remain limited.^16,17 Moreover, the distinct characteristics of CCDC6-RET and NCOA4-RET fusions necessitate further investigation. Therefore, the objective of this study is to determine the predominant RET gene fusions (CCDC6-RET and NCOA4-RET) and analyze their clinicopathological and histological features in a large cohort of Chinese individuals with thyroid cancer.

Materials and Methods

Case identification

This retrospective cohort study collected data from 2300 patients with thyroid cancer who underwent thyroidectomy and next-generation sequencing (NGS) testing at the Fudan University Shanghai Cancer Center from March 2021 to October 2022. Since 2020, NGS has been routinely performed for patients with thyroid cancer undergoing thyroidectomy at our center.

Among the 2300 cases assessed, 2234 were PTC or differentiated high-grade thyroid carcinoma (DHGTC), 24 were medullary thyroid cancer (MTC), 35 were follicular thyroid cancer (FTC), 6 were PDTC, and 1 was ATC. Of these, 113 cases positive for RET fusions were included, with 100 cases of primary tumors confirmed and analyzed and 13 cases of metastasis without primary tumors excluded from this study. Exclusion criteria were as follows: (1) RET fusion-negative (2187 cases) and (2) metastatic tumors without primary tumors in RET fusion-positive (13 cases) (Fig. 1). Clinical and pathological data, including sex, age, primary tumor size, pathological diagnosis, NGS results, presence of distant metastases, thyroid biochemical variables prior to surgical treatment, and date of recurrence, were extracted from electronic medical records.

FIG. 1.

Patient flow diagram. ATC, anaplastic thyroid cancer; DHGTC, differentiated high-grade thyroid carcinoma; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; PDTC, poorly differentiated thyroid cancer; PTC, papillary thyroid cancer.

The clinical stage was determined according to the 8th edition of the American Joint Committee on Cancer TNM staging system for thyroid cancer.¹⁸ Histological evaluation was performed by two experienced pathologists and categorized based on the 5th edition of the World Health Organization (WHO) classification.¹⁹ Moreover, RET fusion tumors were classified into BRAF-like or RAS-like subtypes.¹⁹ Ethical approval for this study was obtained from the Ethics Committee of our hospital (Approval Number: 050432-4-2307E). Informed consent was obtained from each patient for the use of their information in this research.

RNA extraction

The postoperative thyroid tissues containing calcifications were decalcified in ethylenediaminetetraacetic acid solution for 12 hours before being fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Tumor regions were assessed by pathologists using hematoxylin and eosin staining. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples using the QIAamp RNeasy Kit (Qiagen). Detailed RNA quantity and quality information is provided in Supplementary Table S1. RNA concentration, measured using a Qubit 4.0 Fluorometer (Thermo Fisher Scientific), had a median of 36.60 ng/μL (interquartile range: 22.15–58.85 ng/μL), and the median 260/280 ratio, assessed by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), was 1.90 (interquartile range: 1.87–1.95). All procedures followed manufacturer’s instructions.

Multiplex PCR-based library preparation and sequencing

Sequencing libraries were generated using the thyroid cancer multigene panel (RigenBio), specifically designed for amplicon-based NGS. A minimum of 8 ng of RNA was reverse transcribed to complementary DNA and then subjected to two rounds of multiplex PCR amplification, resulting in libraries with unique indexes and adapters. The libraries were purified at various concentrations using magnetic beads (0.8×, 1.2×), quantified with a Qubit 4.0 Fluorometer (Thermo Fisher Scientific), and analyzed for fragment sizes with an Agilent 2100 Bioanalyzer (Agilent Technologies). Subsequently, the qualified libraries underwent paired-end sequencing with 150-bp reads on the NextSeq 550 platform (Illumina). The concentrations of the library are detailed in Supplementary Table S1. The RigenBio thyroid cancer multigene panel enabled the detection of mutations, insertions, deletions, and fusions in a total of 28 genes listed in Supplementary Table S2. All experimental procedures were conducted following the manufacturer’s protocols.

Bioinformatics analysis

Raw sequencing data were processed to remove adapters and low-quality regions using Trimmomatic (version 0.38). The cleaned data were then aligned to the human reference genome (hg19) using the Burrows–Wheeler Aligner tool (version 0.7.10). Single nucleotide variants and insertion/deletion variants (indels) were identified using VarScan (version 2.3.9), whereas gene fusions were detected with FusionMap. Variants and genotype calls were annotated using ANNOVAR and the Variant Effect Predictor. This annotation process included the Sorting Intolerant From Tolerant score, conservation scores such as PolyPhen, and clinically relevant information sourced from ClinVar.

Data analysis

The primary analysis focused on the incidence of RET fusions in Chinese thyroid cancer patients, comparing the distinct clinicopathological features between the CCDC6-RET and NCOA4-RET fusion groups. Hashimoto’s thyroiditis (HT) was diagnosed based on histopathological features, which included interstitial infiltration of hematopoietic mononuclear cells, primarily composed of lymphocytes, plasma cells, and macrophages, as well as the formation of lymphoid follicles.²⁰

The secondary analysis investigated the recurrence rates between the two RET fusion groups, considering the limited availability of follow-up data. In this study, recurrence was defined as the reappearance of structural and biochemical evidence of thyroid cancer in a patient who was initially disease-free postsurgery, necessitating resurgery.²¹ Disease persistence was defined as structural and biochemical residual diseases after the initial surgery.²¹ Patients with persistent thyroid cancer were not considered to have recurred during follow-up.

Statistical analysis

Statistical analysis was performed using Statistical Package for Social Sciences 25.0 (IBM Corporation, USA). The normality of variables was assessed using the Kolmogorov–Smirnov test. Normally distributed continuous variables were presented as mean ± standard deviation, whereas non-normally distributed variables were expressed as medians with interquartile ranges (25th–75th percentiles). Categorical variables were reported as counts and percentages. Continuous variables were compared using Student’s t-test or Mann–Whitney U test, and categorical variables were compared using chi-squared or Fisher’s exact tests. Univariable analyses were performed to compare clinicopathologic and histological features between CCDC6-RET and NCOA4-RET fusions in order to identify independent factors associated with RET fusion. Variables that showed a predetermined statistical difference were then included in multivariable analysis.

To evaluate the risk of disease recurrence associated with RET fusions, we established a control group consisting of a comparable number of cases with the BRAF^V600E mutation and compared the time to disease recurrence between the RET and BRAF groups. In our study, the incidence of the BRAF^V600E mutation was 79.57% (1830/2300), with complete medical records for 1640 primary cases. Propensity score matching was performed to balance baseline variables between the RET (n = 100) and BRAF (n = 1640) groups, using a 1:1 protocol with a caliper width equal to 0.01 of the standard deviation of the logit of the propensity score. Kaplan–Meier survival curve analysis was used to assess cumulative thyroid cancer recurrence based on various fusions or mutations, with differences analyzed using the log-rank test.

Results were presented as odds ratios (OR) with confidence intervals (CI). Bonferroni correction was applied for multiple tests and pairwise comparisons between groups. Statistical significance was set at two-sided p-values <0.05.

Results

Clinicopathological features of the CCDC6-RET and NCOA4-RET groups

Out of 2300 cases of thyroid cancer, RET fusions were exclusively identified in PTC or DHGTC cases (2234 cases), with no presence in other thyroid cancer types (66 cases). Among the 2234 PTC or DHGTC cases, 113 (5.06%) exhibited RET fusions, with 88 (3.94%) showing CCDC6-RET fusion and 25 (1.12%) showing NCOA4-RET fusion. This included 100 primary tumors and 13 metastatic tumors without primary tumors. Specifically, this study analyzed a cohort of 100 cases of primary PTC or DHGTC with RET fusions, where 78.0% (78/100) were CCDC6-RET fusions and 22.0% (22/100) were NCOA4-RET fusions (Fig. 1).

Compared with CCDC6-RET fusion, NCOA4-RET fusion was significantly more prevalent in patients aged ≥45 years (54.5% vs. 28.2%) (p = 0.021) and was associated with a higher incidence of lymph node metastasis (LNM) (90.0% vs. 67.9%) (p = 0.032) (Table 1). Multivariable analysis identified age ≥45 years (OR = 3.916, 95% CI = 1.295–11.843, p = 0.016) and LNM (OR = 6.103, 95% CI = 1.093–34.076, p = 0.039) as independent factors associated with NCOA4-RET fusion (Supplementary Table S3).

Table 1.

Clinicopathologic Features of RET Fusions

Clinicopathologic features	CCDC6-RET fusion	NCOA4-RET fusion	p
Number of patients	78	22
Age, n (%)			0.021
<45 years	56 (71.8)	10 (45.5)
≥45 years	22 (28.2)	12 (54.5)
Female			0.018
<45 years, n (%)	44 (73.3)	5 (35.7)
≥45 years, n (%)	16 (26.7)	9 (64.3)
Male			1.000
<45 years, n (%)	12 (66.7)	5 (62.5)
≥45 years, n (%)	6 (33.3)	3 (37.5)
Tumor size, median (25th–75th percentile) (cm)	1.20 (0.80–2.00)	1.35 (0.60–1.80)	0.656
Multifocality, n (%)			0.553
Yes	15 (19.8)	6 (27.3)
No	63 (80.2)	16 (72.7)
Tumor location in thyroid, n (%)			0.284
Left	39 (50.0)	7 (31.8)
Right	27 (34.6)	12 (54.5)
Isthmus	2 (2.6)	1 (4.5)
Bilateral	10 (12.8)	2 (9.1)
T stage, n (%)			0.130
1a	26 (33.3)	3 (13.6)
1b	16 (20.5)	6 (27.3)
2	8 (10.3)	0 (0.0)
3a	2 (2.6)	0 (0.0)
3b	0 (0.0)	0 (0.0)
4a	13 (16.7)	6 (27.3)
4b	13 (16.7)	7 (31.8)
Clinical N stage, n (%)			0.335
N0	39 (50.0)	8 (36.4)
N1	39 (50.0)	14 (63.6)
Pathological N stage, n (%)			0.024
N0	25 (32.1)	2 (9.1)	0.032*
N1a	32 (41.0)	16 (72.7)	0.009 *
N1b	21 (26.9)	4 (18.2)	0.403*
LNM, n (%)			0.032
Yes	53 (67.9)	20 (90.9)
No	25 (32.1)	2 (9.1)
Number of LNM, n (%)			0.071
1	8 (10.3)	4 (18.2)
≥2	45 (57.7)	16 (72.7)
No	25 (32.1)	2 (9.1)
Local metastases, n (%)			0.364
Yes	4 (5.1)	3 (13.6)
No	74 (94.9)	19 (86.4)
Distant metastases, n (%)	0 (0.0)	0 (0.0)	1.000
Recurrence, n (%)	3 (3.8)	2 (9.1)	0.559

Italicized p-values are statistically significant.

Comparison with other PTC subtypes, and the p-value was adjusted by Bonferroni test. Therefore, p < 0.05/8 was considered as statistically significant.

The staging system used is the 8th edition of the American Joint Committee on Cancer (AJCC) TNM staging system for thyroid cancer (AJCC, 2017).¹⁸ CCDC6, coiled-coil domain containing 6; LNM, lymph node metastasis; N, lymph node status; NCOA4, nuclear receptor coactivator 4; RET, rearranged during transfection; T, primary tumor.

RET fusions and thyroid function indicators

We compared thyroid biochemical variables prior to surgical treatment between the CCDC6-RET and NCOA4-RET groups. Thyroglobulin antibody (TgAb) levels were significantly higher in the CCDC6-RET group compared with the NCOA4-RET group (14.11 [1.86–174.32] IU/mL vs. 2.01 [1.14–15.41] IU/mL) (p = 0.018). In addition, the CCDC6-RET group tended to have higher thyroid peroxidase antibody (TPOAb) levels than the NCOA4-RET group (5.47 [1.00–198.50] IU/mL vs. 1.11 [1.00–8.79] IU/mL), although this difference did not reach statistical significance (p = 0.055). No significant differences were observed in free triiodothyronine (FT3), free thyroxine (FT4), thyrotropin (TSH), or thyroglobulin (Tg) levels between the two groups (Table 2).

Table 2.

Thyroid Function Indicators of the Study Cohort

	CCDC6-RET fusion (n = 78)	NCOA4-RET fusion (n = 22)	p
FT3, mean ± SD (pmol/L)	4.32 ± 0.55	4.59 ± 0.73	0.075
FT4, mean ± SD (pmol/L)	12.66 ± 1.32	12.98 ± 1.87	0.357
TSH, median (25th–75th percentile) (mIU/L)	2.01 (1.22–2.66)	2.25 (1.60–2.98)	0.373
TgAb, median (25th–75th percentile) (IU/mL)	14.11 (1.86–174.32)	2.01 (1.14–15.41)	0.018
TPOAb, median (25th–75th percentile) (IU/mL)	5.47 (1.00–198.50)	1.11 (1.00–8.79)	0.055
Tg, median (25th–75th percentile) (ng/mL)	12.30 (2.50–41.28)	15.65 (8.82–31.70)	0.490

Italicized p-values are statistically significant.

FT3, free triiodothyronine; FT4, free thyroxine; Tg, thyroglobulin; TgAb, thyroglobulin antibody; TPOAb, thyroid peroxidase antibody; TSH, thyrotropin.

Histological features of cases with RET fusions

The distribution of RET fusion among histological types of thyroid cancers is summarized in Supplementary Table S4. The incidence of RET fusions varied among different PTC subtypes: 15.4% (8/52) for diffuse sclerosing PTC (DS-PTC), 7.0% (14/199) for solid PTC, 6.7% (1/15) for clear-cell PTC, 4.0% (52/1286) for classical PTC, 3.2% (14/439) for follicular PTC (FVPTC), and 3.0% (2/66) for oncocytic PTC. In addition, RET fusion was detected in 14.5% (9/62) of DHGTC cases. After analyzing the nuclear findings of PTC, all RET fusion cases were classified as BRAF-like tumors.

Among the 100 primary PTC or DHGTC cases, the CCDC6-RET and NCOA4-RET groups exhibited distinct pathological subtypes. Specifically, CCDC6-RET fusion cases primarily showed classical PTC (56.4%, 44/78) or infiltrative FVPTC (17.9%, 14/78), whereas NCOA4-RET fusion cases were associated with classical PTC (36.4%, 8/22), solid PTC (27.3%, 6/22), or DHGTC (27.3%, 6/22) (Fig. 2). Notably, among DHGTC cases with PTC growth patterns, the prevalence of NCOA4-RET fusion (6 cases) was significantly higher compared with CCDC6-RET fusion (3 cases) (p = 0.003) (Table 3). Multivariable analysis revealed DHGTC (OR = 11.847, 95% CI = 2.184–64.265, p = 0.004) as an independent factor associated with NCOA4-RET fusion (Supplementary Table S3). The CCDC6-RET group exhibited a higher prevalence of HT compared with the NCOA4-RET group (67.9% vs. 22.7%) (p < 0.001) (Table 3). Further details regarding the pathological and morphological characteristics of these subtypes are provided in Supplementary Table S5.

FIG. 2.

Pathological features of primary PTC or DHGTC with RET fusion. (A and B) Classical PTC subtype (×100): The growth pattern consists of papillae with a fibrovascular core lined by neoplastic cells displaying nuclear characteristics, nuclear grooves, and pseudo inclusions (×400). (C and D) Infiltrative FVPTC: Infiltration of follicular growth (×100) with PTC nuclear features (×400). (E and F) DS-PTC (×100): Diffuse infiltration of tumor growth with marked stromal fibrosis, dense lymphocytic infiltration, and numerous psammoma bodies (×200). (G and H) Solid PTC subtype: Solid and nested (insular) growth was observed in the nested areas surrounded by fibrosis (×100). Tumor cells exhibit nuclear features of PTC (×400). (I and J) Oncocytic PTC subtype: Solid growth pattern with PTC nuclei features, abundant oncocytic cytoplasm, and dense red staining (×400). (K and L) Clear-cell PTC subtype: Follicular or solid growth pattern (×100) with small nuclei and pale granular cytoplasm. Mitotic figures are observed in the patient (×400). (M and N) DHGTC: Classical PTC with differentiated areas showing solid, trabecular, or insular growth patterns (×100). High magnification (×400) reveals tumor cells with small nuclei, dispersed chromatin distribution, and visible mitosis (red arrows), lacking PTC nuclear features. DHGTC, differentiated high-grade thyroid carcinoma; DS-PTC, diffuse sclerosing PTC; FVPTC, follicular PTC; RET, rearranged during transfection.

Table 3.

Histological Features of RET Fusions

Histological features	CCDC6-RET fusion	NCOA4-RET fusion	p
Number of patients	78	22
Pathological classification, n (%)			<0.001
Classical PTC	44 (56.4)	8 (36.4)	0.096^*
Infiltrative FVPTC	14 (17.9)	0 (0.0)	0.035^*
DS-PTC	7 (9.0)	1 (4.5)	0.681^*
Solid PTC	8 (10.3)	6 (27.3)	0.075^*
Oncocytic PTC	1 (1.3)	1 (4.5)	0.393^*
Clear-cell PTC	1 (1.3)	0 (0.0)	1.000^*
DHGTC	3 (3.8)	6 (27.3)	0.003 ^*
Calcification, n (%)	59 (75.6)	18 (81.8)	0.543
Gross extrathyroidal extension, n (%)			0.088
Yes	39 (50.0)	16 (72.7)
No	39 (50.0)	6 (27.3)
Surrounding invasion, n (%)			0.265
Yes	14 (17.9)	7 (31.8)
No	64 (82.1)	15 (68.2)
Vascular invasion, n (%)			0.417
Yes	13 (16.7)	7 (31.82)
No	65 (83.3)	15 (68.18)
Perineural invasion, n (%)			0.209
Yes	2 (2.6)	2 (9.1)
No	76 (97.4)	20 (90.9)
Extrathyroidal extension, n (%)			0.070
Yes	26 (33.3)	12 (54.5)
No	52 (66.7)	10 (45.5)
Hashimoto’s thyroiditis, n (%)	53 (67.9)	5 (22.7)	<0.001
Nodular goiter, n (%)	13 (16.7)	6 (27.3)	0.417

Italicized p-values are statistically significant.

Comparison with other PTC subtypes and the p-value was adjusted by Bonferroni test. Therefore, p < 0.05/8 was considered as statistically significant.

DHGTC, differentiated high-grade thyroid carcinoma; DS-PTC, diffuse sclerosing PTC; FVPTC, follicular PTC; PTC, papillary thyroid cancer.

Compound mutations in PTC with RET fusions

In PTC, five cases with RET fusions also exhibited other gene mutations of uncertain significance. These compound mutations included TERT (c.−157T > G, c.−112C > T), CHEK2P2 (n.243T > G), and CTNNB1 (c.2217G > A) in four CCDC6-RET fusion cases and TP53 (c.1120G > A) in one NCOA4-RET fusion case. Notably, cases of RET fusions with compound mutations were associated with older age (≥45 years, 80.0% vs. 20.0%) (p = 0.044) and a higher incidence of bilateral thyroid involvement (60.0% vs. 9.5%) (p = 0.012) compared with those without compound mutations.

Recurrence of PTC or DHGTC with RET fusions

Following propensity score matching, the study included 100 patients with BRAF^V600E mutation. The RET and matched BRAF groups demonstrated similar baseline characteristics postmatching (Supplementary Table S6). The median follow-up periods were 15.2 (6.4–23.2) months for the RET group and 17.0 (6.1–24.0) months for the BRAF group. Specifically, median follow-up periods for the CCDC6-RET and NCOA4-RET groups were 14.1 (4.6–22.2) months and 18.1 (11.0–26.2) months, respectively. Disease persistence was observed in two cases in the CCDC6-RET group, one in the NCOA4-RET group, and three in the BRAF group. No patients with BRAF^V600E mutation experienced recurrence. Among patients with RET fusions, five cases (5.0%, 5/100) experienced recurrence between 7.1 and 21.9 months (Table 4). Specifically, two (9.1%, 2/22) cases with NCOA4-RET fusion and three (3.8%, 3/78) cases with CCDC6-RET fusion experienced recurrence. The RET group exhibited a significantly higher recurrence rate than the BRAF group (5.0% vs. 0.0%, p = 0.018) (Fig. 3). Bonferroni correction (p < 0.05/3) indicated a significantly higher recurrence rate in the NCOA4-RET group (9.1% vs. 0.0%, p = 0.004) compared with the BRAF group but not in the CCDC6-RET group (3.8% vs. 0.0%, p = 0.033). Although the recurrence rate in the NCOA4-RET group was numerically higher than that in the CCDC6-RET group (9.1% vs. 3.8%), there was no statistical difference (p = 0.559) (Fig. 4).

FIG. 3.

Kaplan–Meier curve comparing recurrence rates between RET fusions and BRAF^V600E mutation.

FIG. 4.

Kaplan–Meier curve comparing recurrence rates among CCDC6-RET fusion, NCOA4-RET fusion, and BRAF^V600E mutation.

Table 4.

Clinical Characteristics of Recurrent Cases with RET Fusions

Case #	RET fusion	Age(years)	Tumor location	Tumor size (cm)	Pathology, subtype	TNM stage	I¹³¹ treatment	Follow-up (months)	Recurrence
1	NCOA4	52	L	1.2	DHGTC	I	No	18.27	Yes
17	NCOA4	38	R	0.5	DHGTC	I	No	7.13	Yes
53	CCDC6	28	L	3.5	Classical PTC	I	Yes	10.07	Yes
59	CCDC6	36	B	5.5	DS-PTC	II	No	17.20	Yes
88	CCDC6	59	L	0.8	Classical PTC	I	No	21.93	Yes

B, bilateral thyroid gland; L, left thyroid gland; R, right thyroid gland; TNM, tumor-node-metastasis.

Discussion

RET fusion has been identified as a crucial gene in PTC. Previous research has highlighted that there are no significant differences in the prevalence of RET fusion between Western populations (ranging from 2.50% to 8.70%) and Eastern populations (ranging from 4.35% to 10.81%) with sporadic thyroid cancer.^{7,8,22

–30} However, the incidence of RET fusion notably escalates to 55.0% in pediatric patients with thyroid cancer induced by nuclear exposure.31 Data from The Cancer Genome Atlas (TCGA) database have revealed that RET fusion occurs in 6.80% (33/485) of PTC cases, with CCDC6-RET fusion identified in 4.33% (21/485) and NCOA4-RET fusion in 1.03% (5/485) of cases.³ Our previous study found that the incidence of RET fusion was 7.8% in 217 PTC FFPE samples.³² In the present expanded study involving 2234 PTC or DHGTC FFPE samples, RET fusions were detected in 5.06%, with CCDC6-RET fusions in 3.94% (88/2234) and NCOA4-RET fusions in 1.12% (25/2234) of cases. These findings are consistent with TCGA data and indicate that there is no difference in the incidence of RET fusion between Chinese and Western populations.

Age is an important factor associated with the prevalence of PTC cases with RET fusion.^33,34 Previous studies have indicated a higher prevalence of RET rearrangements in thyroid cancer patients under 45 years old.^28,35 However, differences in age distribution between CCDC6-RET and NCOA4-RET fusions remain unclear. A meta-analysis suggested that children and adolescents exhibit a higher proportion of NCOA4-RET fusions compared with adults.³³ In contrast, our study found a notably higher proportion of patients older than 45 years in the NCOA4-RET group compared with the CCDC6-RET group. In addition, age over 45 years is independently related to NCOA4-RET fusion. Given the limited research in this area, further investigations are warranted.

TgAb and TPOAb levels are essential markers for diagnosing HT, a condition known to increase the risk of developing PTC.^36,37 Elevated TgAb levels (>1150 IU/mL) have been associated with central lymph node metastasis in patients with concurrent PTC and HT.³⁸ However, the differences in TPOAb and TgAb levels between CCDC6-RET and NCOA4-RET fusions have not been extensively studied. Our research found significantly higher TgAb levels in the CCDC6-RET group compared with the NCOA4-RET group. In cases of CCDC6-RET fusion, elevated TgAb levels are associated with a high prevalence of HT. This could be attributed to the following two possible mechanisms: (1) the inflammatory process might facilitate the formation of CCDC6-RET rearrangements or (2) the CCDC6-RET fusion might directly influence the inflammatory response in PTC with HT.^39,40 Further studies are necessary to ascertain whether CCDC6-RET fusion cases with HT and elevated TgAb levels have a better prognosis due to immune system activation.

Previous studies have highlighted that CCDC6-RET fusions frequently occur in classical PTC,⁴¹ infiltrative FVPTC,^42,43 hobnail PTC,^44,45 and DS-PTC,⁴⁶ whereas NCOA4-RET fusions are more commonly found in solid PTC^47,48 and have also been reported in DS-PTC.^49,50 Our investigation revealed that both CCDC6-RET and NCOA4-RET fusions were predominantly detected in classical PTC. Interestingly, CCDC6-RET fusion was more often found in infiltrative FVPTC, whereas NCOA4-RET fusion was frequently observed in solid PTC and DHGTC. Multivariable analysis confirmed an independent association between NCOA4-RET fusion and DHGTC. Importantly, all RET fusions were classified as BRAF-like tumors. According to the 5th WHO classification, DHGTC was categorized as a highly aggressive form of thyroid cancer with a significantly poor 5-year overall survival rate.^50
–52 This implies that NCOA4-RET fusion may contribute to the dedifferentiation of PTC, leading to a poorer prognosis. Given that DHGTC typically shows resistance to radioactive iodine, patients with RET fusions in DHGTC might benefit from treatment with RET kinase inhibitors.^4,53

PTC harboring driver gene fusions, co-occurring with other gene mutations, has been documented to have a higher likelihood of progressing into dedifferentiated and aggressive forms of thyroid cancers.^54,55 RET fusions and BRAF mutations, the most common mutations in PTC, are generally mutually exclusive.²⁶ However, a few reports have documented their co-occurrence in advanced-stage PTC, which is associated with a poor prognosis.^56
–58 In PTC, TERT mutation is a frequent concomitant alteration with RET fusion, occurring in up to 31.11% (14/45) of cases.⁵⁹ RET fusions combined with TERT hotspot mutations (c.−124C>T or c.−146C>T) have been identified as indicators of progression from PTC to PDTC and even to ATC.⁶⁰ In our study, no TERT hotspot or BRAF mutations were detected in cases of PTC or DHGTC with RET fusion. However, we observed a low incidence (5%, 5/100) of compound mutations with uncertain significance. RET fusions with compound mutations were associated with older age (≥45 years) and a higher incidence of bilateral thyroid involvement.

Previous studies have demonstrated that PTC with RET fusions exhibited the highest prevalence of LNM, compared with PTC with BRAF or RAS mutations.⁶¹ Specifically, PTC with RET fusions (involving CCDC6 and NCOA4) was significantly associated with LNM in young adults.⁶² Recent findings suggest that the NCOA4-RET fusion results in a higher incidence of LNM than CCDC6-RET fusion.^24,41 Similarly, our study found a significantly higher incidence of LNM in the NCOA4-RET group compared with the CCDC6-RET group.

In a recent study of 57 patients with PTC over a 250-month follow-up, the recurrence rate did not significantly differ between the CCDC6-RET group and the NCOA4-RET group, with both at 33.33%.²⁴ Our follow-up data indicated an overall recurrence rate of 5% (5/100) for RET fusions. Specifically, the NCOA4-RET group exhibited a higher recurrence rate (9.1%, 2/22) than the CCDC6-RET group (3.8%, 3/78). However, this difference was not statistically significant. Furthermore, comparing recurrence rates between the RET and BRAF groups, a higher recurrence rate was observed in the RET group, suggesting a potentially poorer prognosis. Recurrence rates for the BRAF^V600E mutation have been reported to range from 5.45% to 25.8%.^63,64 However, none of the BRAF^V600E mutation cases in our study experienced recurrence. Given the short follow-up periods for the BRAF^V600E mutated thyroid cancer cases in our study, these conclusions should be interpreted cautiously, and further research is necessary to validate these findings.

Limitations

This study has several limitations, including its inherent retrospective design. This study focuses on RET fusions, omitting other fusion genes like neurotrophic tropomyosin receptor kinase fusion, which have also been reported in thyroid cancer. Moreover, the study’s scope was limited to the two most common RET fusion types (CCDC6-RET and NCOA4-RET), as other RET fusion types were not included in our multigene panel. Furthermore, the low incidence of NCOA4-RET fusion in thyroid cancer resulted in a small sample size, potentially leading to statistical underpowering for some comparisons and analyses. We acknowledge that the sample size did not meet the recommended criteria for multivariable analysis, which typically requires at least 10 events per variable.^65,66 Consequently, caution is warranted in interpreting these findings. In addition, the study was limited by short-term follow-up and a restricted number of cases analyzed for recurrence rates. The survival curve analysis was based on limited data, precluding the conduct of Cox regression analysis. Future investigations should incorporate long-term follow-up to more accurately compare recurrence rates between CCDC6-RET and NCOA4-RET fusions.

Conclusion

This single-center study examined the most common RET fusions in thyroid cancer and provided an in-depth analysis of their histological and clinicopathological characteristics. RET fusions were specifically found in PTC and DHGTC and exhibited distinct clinicopathological features between the CCDC6-RET and NCOA4-RET fusion groups. Notably, the NCOA4-RET group was associated with higher aggressiveness than the CCDC6-RET group. These findings may inform risk stratification of thyroid cancers with RET fusions and highlight the need for timely appropriate treatment.

Footnotes

Authors’ Contributions

Z.W.: Conceptualization, data analysis, methodology, experiment performance, writing—original draft, review, and editing. Q.Y., L.B., H.C., T.X., M.R., Q.W., C.Y., R.W., Y.W., B.P., Q.B.: Experiment performance. Xiaoyan Z.: Conceptualization, funding acquisition, supervision, methodology, writing—original draft, review, and editing. Xiaoli Z.: Conceptualization, funding acquisition, supervision, methodology, writing—original draft, review, and editing.

Author Disclosure Statement

There are no competing financial interests.

Funding Information

This work was supported by the Innovation Program of Shanghai Science and Technology Committee (20Z11900300), the Innovation Group Project of Shanghai Municipal Health Commission Grant (2019CXJQ03), the Shanghai Science and Technology Development Fund (19MC1911000), and the Shanghai Municipal Key Clinical Specialty (shslczdzk01301).

Supplementary Material

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Supplementary Table S5

Supplementary Table S6

References

Romei

, Ciampi

, Elisei

. A comprehensive overview of the role of the RET proto-oncogene in thyroid carcinoma. Nat Rev Endocrinol, 2016; 12(4):192–202; doi: 10.1038/nrendo.2016.11

Santoro

, Carlomagno

. Central role of RET in thyroid cancer. Cold Spring Harb Perspect Biol, 2013; 5(12):a009233; doi: 10.1101/cshperspect.a009233

Cancer Genome Atlas Research Network. Integrated genomic characterization of papillary thyroid carcinoma. Cell, 2014; 159(3):676–690; doi: 10.1016/j.cell.2014.09.050

Salvatore

, Santoro

, Schlumberger

. The importance of the RET gene in thyroid cancer and therapeutic implications. Nat Rev Endocrinol, 2021; 17(5):296–306; doi: 10.1038/s41574-021-00470-9

Romei

, Elisei

. RET/PTC translocations and clinico-pathological features in human papillary thyroid carcinoma. Front Endocrinol (Lausanne), 2012; 3:54; doi: 10.3389/fendo.2012.00054

Nacchio

, Pisapia

, Pepe

, et al. Predictive molecular pathology in metastatic thyroid cancer: The role of RET fusions. Expert Rev Endocrinol Metab, 2022; 17(2):167–178; doi: 10.1080/17446651.2022.2060819

Ullmann

, Thiesmeyer

, Lee

, et al. RET fusion-positive papillary thyroid cancers are associated with a more aggressive phenotype. Ann Surg Oncol, 2022; 29(7):4266–4273; doi: 10.1245/s10434-022-11418-2

Yip

, Nikiforova

, Yoo

, et al. Tumor genotype determines phenotype and disease-related outcomes in thyroid cancer: A study of 1510 patients. Ann Surg, 2015; 262(3):519–525; doi: 10.1097/SLA.0000000000001420

Prescott

, Zeiger

. The RET oncogene in papillary thyroid carcinoma. Cancer, 2015; 121(13):2137–2146; doi: 10.1002/cncr.29044

10.

Staubitz

, Musholt

, Schad

, et al. ANKRD26-RET—A novel gene fusion involving RET in papillary thyroid carcinoma. Cancer Genet, 2019; 238:10–17; doi: 10.1016/j.cancergen.2019.07.002

11.

Toda

, Yamamoto

, Ookubo

, et al. Lenvatinib and selpercatinib successfully treated RET fusion gene-positive papillary thyroid carcinoma cardiac metastases: A case report. Gland Surg, 2023; 12(10):1441–1448; doi: 10.21037/gs-23-252

12.

Yakushina

, Lerner

, Lavrov

. Gene fusions in thyroid cancer. Thyroid, 2018; 28(2):158–167; doi: 10.1089/thy.2017.0318

13.

, McCusker

, Russo

, et al. RET fusions in solid tumors. Cancer Treat Rev, 2019; 81:101911; doi: 10.1016/j.ctrv.2019.101911

14.

Smida

, Salassidis

, Hieber

, et al. Distinct frequency of ret rearrangements in papillary thyroid carcinomas of children and adults from Belarus. Int J Cancer, 1999; 80(1):32–38; doi: 10.1002/(sici)1097-0215(19990105)80:1<32::aid-ijc7>3.0.co;2-l

15.

Sugg

, Ezzat

, Zheng

, et al. Oncogene profile of papillary thyroid carcinoma. Surgery, 1999; 125(1):46–52; doi: 10.1016/S0039-6060(99)70287-4

16.

Mayr

, Pötter

, Goretzki

, et al. Expression of ret/PTC1, -2, -3, -delta3 and -4 in German papillary thyroid carcinoma. Br J Cancer, 1998; 77(6):903–906; doi: 10.1038/bjc.1998.149

17.

Prasad

, Vyas

, Horne

, et al. NTRK fusion oncogenes in pediatric papillary thyroid carcinoma in northeast United States. Cancer, 2016; 122(7):1097–1107; doi: 10.1002/cncr.29887

18.

Tuttle

, Haugen

, Perrier

. Updated American joint committee on cancer/tumor-node-metastasis staging system for differentiated and anaplastic thyroid cancer (Eighth Edition): What changed and why? Thyroid, 2017; 27(6):751–756; doi: 10.1089/thy.2017.0102

19.

WHO Classification of Tumours Editorial Board. Endocrine and neuroendocrine tumours. International Agency for Research on Cancer; 2022. Available from: https://tumourclassification.iarc.who.int [Last accessed: July 10, 2024 ].

20.

Caturegli

, De Remigis

, Rose

. Hashimoto thyroiditis: Clinical and diagnostic criteria. Autoimmun Rev, 2014; 13(4–5):391–397; doi: 10.1016/j.autrev.2014.01.007

21.

de Castro

, Waissmann

, Simões

, et al. Predictors for papillary thyroid cancer persistence and recurrence: A retrospective analysis with a 10-year follow-up cohort study. Clin Endocrinol (Oxf), 2016; 85(3):466–474; doi: 10.1111/cen.13032

22.

Bulanova Pekova

, Sykorova

, Mastnikova

, et al. RET fusion genes in pediatric and adult thyroid carcinomas: Cohort characteristics and prognosis. Endocr Relat Cancer, 2023; 30(12):e230117; doi: 10.1530/ERC-23-0117

23.

Yang

, Aypar

, Rosen

, et al. A performance comparison of commonly used assays to detect RET fusions. Clin Cancer Res, 2021; 27(5):1316–1328; doi: 10.1158/1078-0432.CCR-20-3208

24.

Chu

, Wirth

, Farahani

, et al. Clinicopathologic features of kinase fusion-related thyroid carcinomas: An integrative analysis with molecular characterization. Mod Pathol, 2020; 33(12):2458–2472; doi: 10.1038/s41379-020-0638-5

25.

, Zhang

, Feng

, et al. Targeted next generation sequencing identifies somatic mutations and gene fusions in papillary thyroid carcinoma. Oncotarget, 2017; 8(28):45784–45792; doi: 10.18632/oncotarget.17412

26.

Liang

, Cai

, Feng

, et al. Genetic landscape of papillary thyroid carcinoma in the Chinese population. J Pathol, 2018; 244(2):215–226; doi: 10.1002/path.5005

27.

Shi

, Wang

, Zhang

, et al. Identification of RET fusions in a Chinese multicancer retrospective analysis by next-generation sequencing. Cancer Sci, 2022; 113(1):308–318; doi: 10.1111/cas.15181

28.

Wang

, Tang

, Hua

, et al. Genetic and clinicopathologic characteristics of papillary thyroid carcinoma in the chinese population: High BRAF mutation allele frequency, multiple driver gene mutations, and RET fusion may indicate more advanced TN stage. Onco Targets Ther, 2022; 15:147–157; doi: 10.2147/OTT.S339114

29.

Toda

, Iwasaki

, Okubo

, et al. The frequency of mutations in advanced thyroid cancer in Japan: A single-center study. Endocr J, 2024; 71(1):31–37; doi: 10.1507/endocrj.EJ23-0342

30.

Okubo

, Toda

, Kadoya

, et al. Clinicopathological analysis of thyroid carcinomas with the RET and NTRK fusion genes: Characterization for genetic analysis. Virchows Arch, 2024:1–10; doi: 10.1007/s00428-024-03777-w

31.

de Sousa

MSA

, Nunes

, Christiano

, et al. Genetic alterations landscape in paediatric thyroid tumours and/or differentiated thyroid cancer: Systematic review. Rev Endocr Metab Disord, 2024; 25(1):35–51; doi: 10.1007/s11154-023-09840-2

32.

Ren

, Yao

, Bao

, et al. Diagnostic performance of next-generation sequencing and genetic profiling in thyroid nodules from a single center in China. Eur Thyroid J, 2022; 11(3):e210124; doi: 10.1530/ETJ-21-0124

33.

, Li

, He

, et al. Radiation exposure, young age, and female gender are associated with high prevalence of RET/PTC1 and RET/PTC3 in papillary thyroid cancer: A meta-analysis. Oncotarget, 2016; 7(13):16716–16730; doi: 10.18632/oncotarget.7574

34.

Lee

, Lee

, Im

, et al. NTRK and RET fusion-directed therapy in pediatric thyroid cancer yields a tumor response and radioiodine uptake. J Clin Invest, 2021; 131(18):e144847; doi: 10.1172/JCI144847

35.

, Jia

, Qian

, et al. Genomic characterization of high-recurrence risk papillary thyroid carcinoma in a southern Chinese population. Diagn Pathol, 2020; 15(1):49; doi: 10.1186/s13000-020-00962-8

36.

Boi

, Pani

, Mariotti

. Thyroid autoimmunity and thyroid cancer: Review focused on cytological studies. Eur Thyroid J, 2017; 6(4):178–186; doi: 10.1159/000468928

37.

Spencer

. Clinical review: Clinical utility of thyroglobulin antibody (TgAb) measurements for patients with Differentiated Thyroid Cancers (DTC). J Clin Endocrinol Metab, 2011; 96(12):3615–3627; doi: 10.1210/jc.2011-1740

38.

Min

, Huang

, Wei

, et al. Preoperatively predicting the central lymph node metastasis for papillary thyroid cancer patients with Hashimoto’s thyroiditis. Front Endocrinol (Lausanne), 2021; 12:713475; doi: 10.3389/fendo.2021.713475

39.

Rhoden

, Unger

, Salvatore

, et al. RET/papillary thyroid cancer rearrangement in nonneoplastic thyrocytes: Follicular cells of Hashimoto’s thyroiditis share low-level recombination events with a subset of papillary carcinoma. J Clin Endocrinol Metab, 2006; 91(6):2414–2423; doi: 10.1210/jc.2006-0240

40.

Fusco

, Santoro

. 20 years of RET/PTC in thyroid cancer: Clinico-pathological correlations. Arq Bras Endocrinol Metabol, 2007; 51(5):731–735; doi: 10.1590/s0004-27302007000500010

41.

Khonrak

, Watcharadetwittaya

, Chamgramol

, et al. RET rearrangements are relevant to histopathologic subtypes and clinicopathological features in Thai papillary thyroid carcinoma patients. Pathol Oncol Res, 2023; 29:1611138; doi: 10.3389/pore.2023.1611138

42.

de Vries

, Celestino

, Castro

, et al. RET/PTC rearrangement is prevalent in follicular Hürthle cell carcinomas. Histopathology, 2012; 61(5):833–843; doi: 10.1111/j.1365-2559.2012.04276.x

43.

Celestino

, Sigstad

, Løvf

, et al. Survey of 548 oncogenic fusion transcripts in thyroid tumors supports the importance of the already established thyroid fusions genes. Genes Chromosomes Cancer, 2012; 51(12):1154–1164; doi: 10.1002/gcc.22003

44.

Poma

, Macerola

, Proietti

, et al. Clinical-pathological features and treatment outcome of patients with hobnail variant papillary thyroid carcinoma. Front Endocrinol (Lausanne), 2022; 13:842424; doi: 10.3389/fendo.2022.842424

45.

Lubitz

, Economopoulos

, Pawlak

, et al. Hobnail variant of papillary thyroid carcinoma: An institutional case series and molecular profile. Thyroid, 2014; 24(6):958–965; doi: 10.1089/thy.2013.0573

46.

Chou

, Qiu

, Crayton

, et al. A detailed histologic and molecular assessment of the diffuse sclerosing variant of papillary thyroid carcinoma. Mod Pathol, 2023; 36(12):100329; doi: 10.1016/j.modpat.2023.100329

47.

Kure

, Wada

, Naito

. Relationship between genetic alterations and clinicopathological characteristics of papillary thyroid carcinoma. Med Mol Morphol, 2019; 52(4):181–186; doi: 10.1007/s00795-019-00217-6

48.

Acquaviva

, Visani

, Repaci

, et al. Molecular pathology of thyroid tumours of follicular cells: A review of genetic alterations and their clinicopathological relevance. Histopathology, 2018; 72(1):6–31; doi: 10.1111/his.13380

49.

Alswailem

, Alghamdi

, Alotaibi

, et al. Molecular genetics of diffuse sclerosing papillary thyroid cancer. J Clin Endocrinol Metab, 2023; 108(9):e704–e711; doi: 10.1210/clinem/dgad185

50.

Chiba

. Molecular pathology of thyroid tumors: Essential points to comprehend regarding the latest WHO classification. Biomedicines, 2024; 12(4):712; doi: 10.3390/biomedicines12040712

51.

Dettmer

, Schmitt

, Steinert

, et al. Poorly differentiated thyroid carcinomas: How much poorly differentiated is needed? Am J Surg Pathol, 2011; 35(12):1866–1872; doi: 10.1097/PAS.0b013e31822cf962

52.

Bichoo

, Mishra

, Kumari

, et al. Poorly differentiated thyroid carcinoma and poorly differentiated area in differentiated thyroid carcinoma: Is there any difference? Langenbecks Arch Surg, 2019; 404(1):45–53; doi: 10.1007/s00423-019-01753-6

53.

Ibrahimpasic

, Ghossein

, Shah

, et al. Poorly differentiated carcinoma of the thyroid gland: Current status and future prospects. Thyroid, 2019; 29(3):311–321; doi: 10.1089/thy.2018.0509

54.

Yoo

, Song

, Lee

, et al. Integrative analysis of genomic and transcriptomic characteristics associated with progression of aggressive thyroid cancer. Nat Commun, 2019; 10(1):2764; doi: 10.1038/s41467-019-10680-5

55.

Nguyen

, Fong

, Luthra

, et al. Genomic characterization of metastatic patterns from prospective clinical sequencing of 25,000 patients. Cell, 2022; 185(3):563–575.e11; doi: 10.1016/j.cell.2022.01.003

56.

Henderson

, Shellenberger

, Williams

, et al. High rate of BRAF and RET/PTC dual mutations associated with recurrent papillary thyroid carcinoma. Clin Cancer Res, 2009; 15(2):485–491; doi: 10.1158/1078-0432.CCR-08-0933

57.

Costa

, Herrero

, Fresno

, et al. BRAF mutation associated with other genetic events identifies a subset of aggressive papillary thyroid carcinoma. Clin Endocrinol (Oxf), 2008; 68(4):618–634; doi: 10.1111/j.1365-2265.2007.03077.x

58.

Zou

, Baitei

, Alzahrani

, et al. Concomitant RAS, RET/PTC, or BRAF mutations in advanced stage of papillary thyroid carcinoma. Thyroid, 2014; 24(8):1256–1266; doi: 10.1089/thy.2013.0610

59.

Zhang

, Lin

, Wang

, et al. Coexisting RET/PTC and TERT promoter mutation predict poor prognosis but effective RET and MEK targets in thyroid cancer. J Clin Endocrinol Metab, 2024; doi: 10.1210/clinem/dgae327

60.

Landa

, Cabanillas

. Genomic alterations in thyroid cancer: Biological and clinical insights. Nat Rev Endocrinol, 2024; 20(2):93–110; doi: 10.1038/s41574-023-00920-6

61.

Adeniran

, Zhu

, Gandhi

, et al. Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas. Am J Surg Pathol, 2006; 30(2):216–222; doi: 10.1097/01.pas.0000176432.73455.1b

62.

Sassolas

, Hafdi-Nejjari

, Ferraro

, et al. Oncogenic alterations in papillary thyroid cancers of young patients. Thyroid, 2012; 22(1):17–26; doi: 10.1089/thy.2011.0215

63.

Huang

, Gao

, Bian

, et al. Associations of BRAF V600E, clinical pathology and imaging factors with the recurrence rate of papillary thyroid microcarcinoma. Exp Ther Med, 2020; 20(6):243; doi: 10.3892/etm.2020.9373

64.

Xing

, Alzahrani

, Carson

, et al. Association between BRAF V600E mutation and recurrence of papillary thyroid cancer. J Clin Oncol, 2015; 33(1):42–50; doi: 10.1200/JCO.2014.56.8253

65.

Peduzzi

, Concato

, Feinstein

, et al. Importance of events per independent variable in proportional hazards regression analysis. II. Accuracy and precision of regression estimates. J Clin Epidemiol, 1995; 48(12):1503–1510; doi: 10.1016/0895-4356(95)00048-8

66.

Peduzzi

, Concato

, Kemper

, et al. A simulation study of the number of events per variable in logistic regression analysis. J Clin Epidemiol, 1996; 49(12):1373–1379; doi: 10.1016/s0895-4356(96)00236-3