Due to the increased application of ultrasound and thyroid fine needle aspiration (FNA) biopsy, thyroid cancer (TC), a tumor originating from the thyroid follicular or parafollicular epithelium, has an increasing incidence and becomes the most common endocrine malignant tumor (1–3). Non-medullary thyroid carcinoma (NMTC) arises from thyroid follicular epithelial cells and includes papillary, follicular, and undifferentiated carcinomas, accounting for 95% of TC. NMTC, which mostly occurs in a sporadic fashion, usually has less aggressive behavior and a good prognosis (4).

Familial non-medullary thyroid carcinoma (FNMTC), consisting of 5–10% of thyroid carcinoma, is defined as NMTC occurring in two or more first-degree relatives in the absence of other predisposing causes of thyroid cancer (3–6). There has been debates about the difference in aggressiveness between FNMTC and sporadic non-medullary thyroid carcinoma (SNMTC). Several studies reported that FNMTC tended to be more aggressive than SNMTC, and FNMTC had increased risk of multifocal, bilateral growth, capsular and vascular invasion, and lymph node metastasis, with relatively short disease-free survival (5, 7–10). The patients with three or more cases in a family were more aggressive and had a poorer prognosis than those with only two cases in a family (7), and parent–offspring FNMTC might have more aggressive biological behavior and a worse prognosis than sibling FNMTC (11). However, there were other studies indicated that FNMTC was no more aggressive than SNMTC (12–15).

v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common genetic mutation in papillary thyroid carcinoma (PTC); it is associated with thyroid cancer by activating the mitogen-activated protein kinase pathway (16). Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase. The two most common mutations in the TERT promoter region are C228T and C250T, which can enhance the transcriptional activity of the TERT promoter and are frequently found in TC (17). At present, some studies have performed combined analysis of BRAF V600E mutation and TERT promoter mutation in NMTC and found that these mutations were significantly related to the pathogenesis, development, and prognosis of NMTC (18, 19). Some studies compared the type of common gene mutations, including BRAF, Ras, between FNMTC and SNMTC, and found no significant differences (13).

To clarify the clinicopathological characteristics of FNMTC and compare the tumor aggressiveness between these two malignancies, we retrospectively analyzed the PTC patients who underwent thyroid FNA in West China Hospital.

We reviewed 177 patients from 80 families who underwent thyroid FNA and were diagnosed with NMTC in West China Hospital, Sichuan University, during 2011–2019. Those patients including 175 PTC and 2 follicular carcinoma had no other predisposing causes of thyroid cancer and had been classified as FNMTC according to criteria defined by Sturgeon (20).

However, only 106 patients with postoperative pathology diagnosis of PTC were included in this study due to the incomplete clinicopathological data caused by the fact that some patients did not receive surgery in our hospital (including 5 patients who did not receive surgery, and the rest were operated on in other hospitals), and some patients were lost to follow-up. A total of 7,663 patients were diagnosed with PTC in our hospital records system, and 212 cases were randomly selected to match 1:2 ratio for study purpose. All of those patients’ negative family history of thyroid carcinoma was confirmed during the telephone follow-up. The mean follow-up time for FNMTC patients was 42 months (6–120 months) and that for SNMTC was 44.5 months (7–102 months).

T (the extent of the primary tumor), N (regional lymph-node metastases), and M (distant metastases) staging was determined according to the eighth edition of the American Joint Committee on Cancer (AJCC) thyroid cancer staging criteria.

DNA from paraffin-embedded tumor tissues (all samples came from preoperative FNA cell blocks or postoperative tumor tissue paraffin blocks) was extracted according to the instructions of QIAamp DNA FFPE Tissue Kit from QIAGEN, Germany. The fC-1100 ultrafine ultraviolet spectrophotometer produced by Hangzhou Suizhen Co., Ltd. was used to detect its purity and concentration and stored at −20°C for future use.

Polymerase chain reaction (PCR) was used for amplification. For BRAF V600E, PCR primers were used to amplify the exon 15 of BRAF gene containing mutation hotspots (16). For TERT promoter mutations, the previously established PCR primers 50-AGTGGATTCGCGGGCACAGA-30 (sense) and 50-CAGCGCTGCCTGAAACTC-30 (antisense) were used to amplify the TERT promoters containing two mutation hotspots (C228T and C250T) (17). These materials were carried out with an initial denaturation step at 95°C for 3 min, followed by 10 cycles of 95°C denaturation for 30 s, 55°C annealing for 30s, and 68°C elongation for 1 min. Before sequencing, 20 g/L agarose gel electrophoresis was used to detect the quality of PCR amplification products, and Sanger sequencing was performed on PCR amplification products with satisfactory quality. The sequencing results were compared with the BRAF and TERT gene sequences to confirm the mutation status.

With up to 10% of NMTC (3–6), FNMTC was generally considered as a separate malignancy in thyroid cancer with controversial clinical behavior and prognosis. At present, several studies have shown that FNMTC was more aggressive and had a worse prognosis than SNMTC (5, 7–11), but some studies have shown that there was no significant difference in biological behavior or prognosis between the two diseases (12–14). Since lots of medical records did not demonstrate whether the patient had a family history, according to the data we collected, FNMTC cases accounted for nearly 2% in our research, lower than those of former studies. We decided to randomly selected 212 patients without family history to match 1:2 ratio. However, due to limited sample data, age, gender, and tumor stage were not matched.

Cao et al. (10) and Ito et al. (15) found that FNMTC had higher multiple foci incidence, and Cao’s study also showed that it has a higher bilateral incidence. Zhang et al. found a higher incidence of lymph nodes metastases of FNMTC (5). Meanwhile, compared with SNMTC, FNMTC had a higher recurrence rate and mortality (7, 10, 21, 22). However, Moses et al. (13) found that there were no significant differences between FNMTC and SNMTC in multiple foci, bilateral incidence, and the rate of lateral lymph node metastasis; Ito et al. and Zhang et al. also proved that there was no significant difference in recurrence mortality between the two types.

Recently, with the extensive use of thyroid ultrasound technology and FNA in China, TC was found and treated at an early stage. In our study, we compared some of the invasion-related risk factors, including gender, age, tumor size, capsular invasion, bilateral growth, with thyroid nodular goiter or not, multifocality, lymph node metastasis, extrathyroidal extension, iodine 131 treatment, T stage, and AJCC stage, for FNMTC and SNMTC. We found that the tumor size of FNMTC is significantly smaller than SNMTC (p = 0.020), while there was no significant difference in the incidence of other factors (p > 0.05). We also compared the recurrence rate and mortality between FNMTC and SNMTC; same with Moses et al. and Ito et al, there was no significant difference between these two types (p > 0.05). One hundred four FNMTC patients (98.11%) and 208 (98.11%) SNMTC patients belong to AJCC I and II stage. These results suggested that the aggressiveness of FNMTC was not significantly different from that of SNMTC, while the average tumor size of our data (0.96 and 1.15 cm) was smaller than that of Park et al. (11) (1.2 and 1.4 cm), Robenshtok et al. (14) (1.78 and 2.02 cm), and Uchino et al. (22) (1.98 and 2.05 cm). These might mean that NMTC was detected early with thyroid ultrasound technology and FNA, and FNMTC was detected earlier than SNMTC.

Another study by Zhang et al. (7) showed that the subgroup with three or more members in the family affected with the disease was more invasive than the subgroup with only two members infected. Park et al. (11) also believed that the parent–child group was more aggressive than the sibling group, while Cao et al. (10) came to a different conclusion. Therefore, we also compared the invasive differences between different subgroups and SNMTC patients. Similar to Cao et al. (10), SNMTC only showed more significant opportunity with Hashimoto thyroiditis than two affected members group (p = 0.046). This could be explained by the research conclusions of Zeng et al. and Azizi et al.; they suggested that Hashimoto’s thyroiditis played a protective factor in NMTC (23, 24). Our results showed that there were no significant statistical differences in other factors between each subgroup and between each subgroup and SNMTC (p > 0.05), i.e., the aggressiveness of each subgroup was no more than that of SNMTC. However, the number of patients from three or more person families was relatively small, and further studies are needed to compare.

These results might be caused by the early detection and intervention of FNMTC when the tumor size of FNMTC remains small. We speculated that due to the extensive application of ultrasound and FNA in China and the active monitoring of asymptomatic first-degree relatives of patients with FNMTC, it can be detected early.

Studies have shown that BRAF V600E mutation was associated with large tumor size, thyroid capsule invasion, extraglandular invasion, lymph node metastasis, and high AJCC stage in PTC patients (25, 26). The TERT promoter mutation was related to the age of PTC patients, the maximum tumor diameter, the status of thyroid capsular invasion, and AJCC stage, according to Jin et al. (27) and Alzahrani et al. (28). Unfortunately, similar results were not obtained in our current study, which may be related to the small sample size of our genetic testing, and TERT was not analyzed due to the small number of mutations. Moreover, in one of our previous studies (29), we found that BRAF V600E mutations were mainly associated with recurrence and metastasis, which also confirmed that BRAF could be used as an indicator to evaluate the aggressiveness of NMTC. Therefore, BRAF V600E and TERT promoter mutations were also used as the evaluation indicators of FNMTC aggressiveness and was compared with SNMTC; the difference between them was not statistically significant (p > 0.05). This also indicated that there was no difference in clinicopathological invasiveness between FNMTC and SNMTC from a genetic perspective, while researchers generally believed that BRAF V600E can drive the growth of PTC through the mitogen-activated protein kinase (MAPK) pathway, and TERT promoter mutation might have a similar effect (25–30).

There were some limitations in our study. First, only 106 patients with FNMTC were finally included in the comparison of clinicopathological features. In the subgroup analysis, the case number of some subgroups was small, so the comparison could not be made in the statistical analysis, and the comparison results were meaningless. Furthermore, the biological behavior of NMTC patients was less aggressive, and the prognosis was better than that of other malignant tumors, so long-term follow-up is needed to explore the recurrence and metastasis. The average follow-up time of this study was 3–4 years, and some patients still need to be followed up to evaluate the recurrence and metastasis. All the patients in this study received surgical treatment shortly after the diagnosis of NMTC by FNA, so it was impossible to compare the biological behavior and prognosis differences between early and late intervention of FNMTC in this study, which need further exploration and study by follow-up researchers.

In summary, by comparing the biological behavior, prognosis, and molecular level of FNMTC and SNMTC, we draw conclusion that the biological behavior and prognosis of FNMTC were no more aggressive and worse than SNMTC, and BRAF V600E and TERT also provided a genetic explanation for this conclusion. We speculated that it might be the early detection of FNMTC with increasing emphasis on the family history that led to the result of a significantly smaller mean tumor size of FNMTC than that of SNMTC.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

All procedures in this study were reviewed and approved by the Clinical Trial and Biomedical Ethics Committee of West China Hospital of Sichuan University.

TY was responsible for the experimental design, experimental data collection, analysis and drafting of papers. LH assisted in data collection and analysis. CC was mainly responsible for genetic testing of samples. HL was responsible for experimental guidance and assisted in data collection. YJ was responsible for the overall experimental design, experimental guidance, and important modification of papers. All authors contributed to the article and approved the submitted version.

This work is supported by Chengdu Science and Technology Program (No. 2019-YF05-00324-SN) and 1·3·5 Project for Disciplines of Excellence–Clinical Research Incubation Project, West China Hospital, Sichuan University (No. 2020HXFH024).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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