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Detection of genetic mutations in 855 cases of papillary thyroid carcinoma by next generation sequencing and its clinicopathological features

Diagnostic Pathology volume 19, Article number: 146 (2024) Cite this article

Abstract

Objective

To investigate the genetic mutations in patients with papillary thyroid carcinoma (PTC) and their clinicopathological features by next generation sequencing (NGS).

Methods

NGS technology was used to detect genetic mutations in PTC patients, and clinicopathological features were collected.

Results

①Among 855 PTC patients, 810 patients had genetic mutations, and 45 patients had no genetic mutation. ②BRAF mutation was associated with tumor diameter (P < 0.001) and histological subtypes (P = 0.002). The abundance of V600E mutation was associated with gender (P = 0.004), tumor diameter (P < 0.001), bilateral presentation (P = 0.001), extrathyroidal extension (P < 0.001), lymphatic metastasis (P < 0.001), histological subtypes (P = 0.002) and TNM staging (P = 0.000); The different mutation abundance of V600E was associated with tumor diameter (P < 0.001), multifocal presentation (P = 0.047), bilateral presentation (P = 0.001), extrathyroidal extension (P = 0.001), lymphatic metastasis (P < 0.001), histological subtypes (P = 0.022) and TNM staging (P = 0.000). ③RET fusion was associated with tumor diameter (P < 0.001) and lymphatic metastasis (P = 0.005). ④TERT mutation was associated with gender (P = 0.043), tumor diameter (P < 0.001), extrathyroidal extension (P = 0.028) and TNM staging (P = 0.017). ⑤RAS mutation was associated with histological subtypes (P < 0.001). ⑥NTRK and PIK3CA mutations were not associated with clinicopathological features.

Conclusion

NGS technology can comprehensively analyze the genetic mutations in PTC patients, which provides important prompts for the occurrence, development, diagnosis and treatment of PTC. In addition, BRAF V600E mutation, RET fusion and TERT mutation are associated with a number of high-risk clinicopathological features. Detection of genetic mutations in PTC patients by NGS is of great significance.

Introduction

Papillary thyroid carcinoma (PTC) is the most common malignant tumor of the endocrine system. In recent decades, the incidence rate of PTC has risen rapidly worldwide, and is expected to become the fourth malignant tumor in the incidence rate within 10 years [1,2,3]. According to histological subtypes, thyroid cancer can be divided into PTC, medullary thyroid cancer, follicular thyroid cancer, and undifferentiated thyroid cancer, with PTC being the most common, accounting for approximately 80–85% [4, 5]. The occurrence of thyroid cancer is closely related to factors such as activation of oncogenes, inactivation of tumor suppressor genes, excessive iodine intake, estrogen, ionizing radiation, and obesity, but the specific mechanisms have not been elucidated [6, 7].

Next Generation Sequencing (NGS) technology has the characteristics of high throughput, high depth and high sensitivity, and has been widely used in the research of neoplastic diseases [8, 9]. In this study, NGS technology was used to clarify the genetic mutations in PTC patients, and to explore the relationship between key genes and clinicopathological features of PTC.

Materials and methods

Clinical data selection

PTC patients in Fujian Medical University Union Hospital from January 2021 to September 2022 were selected. A total of 855 patients were confirmed to have PTC by pathology, and surgical resection samples and clinicopathological features were collected. Among the 855 patients, there were 189 males and 666 females; Age range from 7 to 83 years old, with 665 cases ≤ 55 years old and 190 cases > 55 years old; The diameter of the tumor ranges from 0.1 to 5.0 cm, with 620 cases ≤ 1 cm and 235 cases > 1 cm; 557 patients had single lesion, while 298 patients had multiple lesions; 716 patients were on the single side, and 139 patients were on both sides; 204 patients had absent or incomplete capsule, while while 651 patients had complete of the capsule;401 patients had extrathyroidal extension, while 454 patients had no extrathyroidal extension; 478 patients had lymphatic metastasis, and 377 patients had no lymphatic metastasis; 676 patients had classical subtype, 120 patients had follicular subtype, and 59 patients had both classical subtype and follicular subtype; There were 728 patients with TNM stage I, 110 with stage II, and 7 with stage III. The different histological subtypes of PTC patients are shown in Fig. 1.

NGS sequencing

For paraffin-embedded samples, a nucleic acid extraction kit (DKJ28-01, Guangzhou Meiji Biotechnology Co., Ltd.) was utilized for extraction. The concentration was detected by employing the gene sequencing universal library kit (dsDNA quantification) (230228C03Z, Xiamen Aide Biological Company) and the Quantus fluorescence photometer (E6150, Peomega Company, USA). The human tumor multi-gene mutation detection kit (reversible end termination sequencing) (220701C01Z, Xiamen Aide Biological Company) was used to construct a library. DNA fragmentation was accomplished through enzyme digestion, followed by end repair, splicing, and PCR amplification to obtain an amplified library. The amplified library was subjected to liquid-phase hybridization with probes, capture enrichment via the magnetic bead method, and PCR amplification to yield the captured library. Quality control of pre-library and captured library fragments was conducted using the 2100 Bioanalyzer System (Agilent Technologies, Germany). Sequencing was performed using the Illumina MiseqDx sequencer (Illumina, USA). After filtering the raw sequencing data, the Aide Bioinformatics Analysis System was adopted for bioinformatics analysis. Using GRCh37/hg19 as the reference sequence, analyze and detect all hot exon regions and some introns of human AKT1, ALK, BRAF, CTNNB1, EGFR, EIF1AX, FGFR1, FGFR2, FGFR3, FGFR4, GNAS, HRAS, KDR, KIT, KRAS, MET, NRAS, NTRK1, NTRK2, NTRK3, PAX8, PDGFRA, PIK3CA, PTEN, RASAL1, RET, TERT, TP53, TSC2, TSHR; The types of genetic mutations detected include point mutation, insertion deletion variation, fusion, copy number variation, etc.

Genetic mutations with an abundance of 1% or more were considered positive. For hot spot mutation below 1%, PCR validation was required. The primer sequence for BRAF V600E mutation and internal reference are shown in Table 1. The methodology of PCR technology is as follows. After nucleic acid extraction, DNA was obtained from tissue samples. A PCR reaction system was established according to the kit instructions. Then PCR amplification is performed on a fluorescence quantitative PCR instrument. Finally, the results were interpreted to complete the validation.

Table 1 Primer sequence of PCR
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Bioinformatics analysis

The following analysis should be carried out for the detection data: ①Data quality control: Perform quality control on the data obtained through sequencing to eliminate low-quality sequences and possible contaminated data. ②Data alignment: Align the sequencing data with the reference genome to determine the position of each sequencing fragment on the genome. ③Sorting and deduplication: After comparison, sort the sequence and remove duplicate sequences to enhance the accuracy of subsequent analysis. ④Obtaining BAM file: After the above steps, a BAM file was generated, which contains the information of sequencing data and reference genome alignment. ⑤Annotation and filtering: Annotate the test results to determine gene, location and other information, and filter to remove false positive results to obtain the results.

This study only detected somatic variants of tumors and did not conduct germline gene detection using peripheral blood. The variants information obtained after sequencing were compared with public databases such as 1000 Genomes (http://www.internationalgenome.org/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), COSMIC (http://cancer.sanger.ac.uk) to rule out the possibility of germline mutation. Simultaneously, the Aide Bioinformatics Analysis System we utilize contains a white blood cell database which can assist us in screening for SNP loci, benign SNP loci can be removed.

Variation classification

Referring to the “Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer” by the Association for Molecular Pathology (AMP), American Society of Clinical Oncology (ASCO), College of American Pathologists (CAP), by integrating the relevant research evidence of variants in treatment, diagnosis, and prognosis, the variants were divided into: Tier I (variants with strong clinical significance), Tier II (variants with potential clinical significance), Tier III (variants with unknown clinical significance), and Tier IV (variants deemed benign or likely benign). In this study, variants of Tier I and Tier II were included [10].

Statistical analysis

The experimental data was analyzed by SPSS 26.0 statistical software. The chi-square test was employed to compare the counting data. Specifically, when the total sample size n ≥ 40 and the theoretical frequencies of all cells n ≥ 5, the Pearson chi-square test was utilized; In cases where n ≥ 40 but some theoretical frequencies n<5, a continuity correction test was applied. For sample sizes n<40 or theoretical frequencies n<1, Fisher’s exact probability method should be used. Comparison of measurement data was conducted using a T-test with statistical significance defined as P < 0.05.

Results

Among the 855 PTC patients, 810 patients were found to have genetic mutations, while 45 patients were found to have no genetic mutation. Among the 810 patients with genetic mutations, 775 patients had single genetic mutation, while 35 had two genetic mutations. The highest mutation rate was for the BRAF (n = 743), with a mutation rate of 86.90%. The other genes in order of mutation rate were RET (n = 43), KRAS (n = 10), TERT (n = 9), NTRK3 (n = 8), NRAS (n = 7), TP53 (n = 5), PIK3CA (n = 7), NTRK1 (n = 4) and ALK (n = 2) etc. The detail of genetic mutations are shown in Table 2.

Table 2 Genetic mutations in PTC
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Fig. 1
figure 1

(1-1: classical subtype; 1-2: follicular subtype; 1-3: classical and follicular subtype)

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BRAF

Among the 743 BRAF mutation, there were 732 cases of V600E mutation, 1 case of V600-K601delinsE mutation, 1 case of V601E mutation, and 9 fusion (with IGF2BP2, OSBPL9, WARS1, MKRN1, KATNA1, MBP, B1CD2, MBNL2, TRIM24, respectively). In addition, 33 cases of V600E mutation patients had co-mutation with other genes, including AKT1, EGFR, E1FIAX, KRAS, NRAS, HRAS, NTRK1, TP53, PIK3CA, TERT and FGFR3 (Table 2). We divided 855 cases of PTC patients into the BRAF mutation group (n = 743) and the BRAF wild-type group (n = 112), and then compared the clinicopathological features in the two groups. The results showed that the BRAF mutation was only associated with tumor diameter (P < 0.001) and histological subtypes (P = 0.002) (Table 3). Furthermore, we divided PTC patients into the V600E mutation group and the V600E wild-type group, the result was consistent with above.

Table 3 Relationship between BRAF/RET/TERT mutations and clinicopathology features of PTC, while the clinicopathology features of RAS/NTRK/PIK3CA mutations in PTC
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To further investigate the relationship between the abundance of V600E mutation and clinicopathological features, V600E mutation patients were divided into different groups according to their clinicopathological features. The results showed that the abundance of V600E mutation was related to gender(P = 0.004), tumor diameter(P < 0.001), bilateral presentation(P = 0.001), extrathyroidal extension (P < 0.001), lymphatic metastasis(P < 0.001), histological subtypes(P = 0.002), and TNM staging (P = 0.000) (Fig. 2).

In 732 patients with V600E mutation, the mutation abundance ranged from 0.09 to 38.93%, and 26 cases of PTC patients with mutation abundance below 1% were verified by PCR. According to the 20% mutation abundance as the limit, 732 patients were divided into low mutation abundance group (abundance ≤ 20%) and high mutation abundance group (abundance > 20%), and the differences of clinicopathological features in each group were compared. The results showed that the different abundance of V600E mutation was correlated with the tumor diameter(P < 0.001), multifocal presentation(P = 0.047), bilateral presentation(P = 0.001), extrathyroidal extension (P = 0.001), lymphatic metastasis(P < 0.001), histological subtypes(P = 0.022), TNM staging (P = 0.000).

Fig. 2
figure 2

Relationship between the abundance of V600E mutation and the clinicopathological features of PTC

2-1 Gender; 2-2 Age; 2-3 Tumor diameter(cm); 2-4 Multifocal presentation; 2-5 Bilateral presentation; 2-6 Tumor capsule; 2-7 Extrathyroidal extension; 2-8 Lymphatic metastasis; 2-9 Histological subtypes; 2-10 TNM staging

Comparison between groups, *: P < 0.05

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RET

Among the 855 cases, 43 cases of RET fusion were detected, with a mutation rate of 5.03%. Among them, there were 23 cases of CCDC6-RET fusion, 12 cases of NCOA4-RET fusion, 4 cases of ERC1-RET fusion, 2 cases of GOLGA5-RET fusion, 1 case of AFAP1L2-RET fusion, and 1 case of ANKRD26-RET fusion. The clinicopathological features of 43 patients with RET fusion are shown in Table 3.

The PTC patients were divided into RET fusion group (n = 43) and RET wild-type group (n = 812). The differences of clinicopathological features between the two groups were compared. The results showed that RET fusion was associated with tumor diameter (P < 0.001) and lymphatic metastasis (P = 0.005) (Table 3). PTC patients were also divided into CCDC6-RET fusion group (n = 23) and non CCDC6-RET fusion group (n = 832). The results showed that CCDC6-RET fusion was only associated with the tumor diameter (P < 0.001) and lymphatic metastasis (P = 0.029). PTC patients were also divided into the NCOA4-RET fusion group (n = 12) and the non NCOA4-RET fusion group (n = 843). The results show that RET-NCOA4 fusion was only associated with the tumor diameter (P = 0.001).

According to the 20% mutation abundance threshold, the RET fusion patients were divided into low mutation abundance group (n = 31) and high mutation abundance group (n = 12). Comparing the clinicopathological features of different mutation abundance of RET afterward, it was found that different mutation abundance of RET was related to tumor capsule(P = 0.044).

TERT

Among the 855 PTC patients, 9 cases of TERT mutation were detected, with a mutation rate of 1.05%, and all these 9 patients had BRAF V600E co-mutation. In which,7 had the C228T mutation, and 2 had the C250T mutation. The clinicopathological features of TERT patients are shown in Table 3.

Comparing the clinicopathological features of the TERT mutation group (n = 9) with TERT wild-type group (n = 846), the results showed that TERT mutation was associated with gender (P = 0.043), tumor diameter (P < 0.001), extrathyroidal extension (P = 0.028), and TNM staging (P = 0.017) (Table 3).

RAS/NTRK/PIK3CA

Among the 855 PTC patients, 18 cases of RAS mutation were detected, with a mutation rate of 2.11%. Among the RAS mutation patients, 10 had KRAS mutation, 7 had NRAS mutation, and 1 had HRAS mutation. 12 cases of NTRK fusion were detected, with a mutation rate of 1.40%. Among the NTRK fusion patients, 4 had NTRK1 fusion, and 8 had NTRK3 fusion. 7 cases of PIK3CA mutation was detected, with a mutation rate of 0.82%. The clinicopathological features of RAS/NTRK/PIK3CA are shown in Table 3.

After comparing the clinicopathological features of the RAS mutation group (n = 18) with RAS wild-type group (n = 837), we found that RAS mutation was only related to histological subtypes (P < 0.001). The same methodology was applied to the comparison of NTRK/PIK3CA and clinicopathological features, and the results showed that NTRK/PIK3CA mutations were not associated with clinicopathological features of PTC patients.

Discussion

NGS has high throughput, high depth, and high sensitivity, and it has been widely used in basic and clinical research. Compared with PCR technology and first-generation sequencing technology, NGS can not only detect common mutations, but also detect rare mutations, which is of great significance [8, 9, 11]. Apart from the commonly observed mutations in PTC, in this study, we have also detected some rare mutations, including fusion and point mutation. The detailed information regarding these rare mutations can be found in Table 2. Applying NGS to the detection of tumor diseases provides important hints and guidance for the occurrence and development of tumor diagnosis and treatment [8, 12]. In this study the most common mutations were found in the BRAF, RET, TERT and RAS. The BRAF, RET and RAS are involved in the mitogen-activated protein kinase (MAPK) signaling pathway, which is the most common carcinogenic mechanism in PTC. The BRAF, RET, and RAS genes usually do not co-mutation, but in our experimental results, we found 6 cases of BRAF with NRAS co-mutation, 1 case of BRAF with KRAS co-mutation, and 1 case of BRAF with HRAS co-mutation. In addition, we detected EGFR, TP53, PIK3CA, TERT, E1FIAX1, FGFR3, AKT1, E1F1AX, and NTRK1 co-mutation with BRAF, these mutations are difficult to be detected by PCR methodology or first-generation sequencing technology.

BRAF was discovered by Ikawa S et al. [13] in neuroblastoma in 1988, and it is located on human chromosome 7 (7q34), encoding a RAF family serine/threonine protein kinase. BRAF plays a role in regulating the MAPK signaling pathway, promoting continuous cell division, proliferation, and tumor formation [14, 15]. The relationship between BRAF and the clinicopathological features of PTC patients has not been clearly established, and different research results vary [16,17,18,19,20,21]. In this study, BRAF mutation was only related to tumor diameter and histological subtypes. Further studies have revealed that the abundance of V600E mutation was associated with gender, tumor diameter, bilateral presentation, extrathyroidal extension, lymphatic metastasis, histological subtypes and TNM staging. Numerous studies had confirmed that BRAF mutation was linked to high-risk clinicopathological features such as lymphatic metastasis, age and extrathyroidal extension (Table 4) [18, 21]. It can be seen that BRAF mutation is an important independent prognostic factor for PTC.

Table 4 Relationship between BRAF/RET/TERT genes and clinicopathological characteristics of PTC patients in different studies
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RET is located on chromosome 10q11.2 of human and was identified as an oncogene through the transfection of human lymphoma DNA into mouse NIH3T3 cells [22]. RET gene encodes the RET protein, which can activate signaling pathways such as RAS, STAT, and PI3K, thereby participating in the proliferation, invasion, and migration of tumor cells [23]. RET fusion account for only 6% of RET variation, but is related to the occurrence, development, and biological behaviors of tumor such as invasion and migration [24]. In this study, RET fusion was detected in 43 patients. The common fusion types among patients with PTC are CCDC6-RET and NCOA4-RET, which is in line with the current consensus as described in references [25, 26]. Presently, there is inconsistency in research findings regarding the correlation between RET and clinicopathological features of PTC patients [27,28,29]. Our investigation revealed an association between RET fusion and tumor diameter as well as lymphatic metastasis, while no significant link existed with other clinicopathological features. Current studies hold that RET fusion is related to factors such as gender, TSH level, lymphatic metastasis, immune microenvironment, progressive histopathological features, later T stages, and patients with RET fusion are more likely to experience recurrence [27,28,29] (Table 4).

TERT is the catalytic protein subunit of telomerase. The abnormal activation of TERT in telomeres is of great significance to the biological behavior of tumor cells, such as proliferation, invasion and migration [30]. The most common mutations are C228T and C250T. The mutation of these two sites will produce a new set site E-26 transcription factor, thus promoting the transcriptional activity of telomerase [31]. The results of this study showed that TERT mutation was related to gender, tumor diameter, extrathyroidal extension and TNM stating. Current research indicates that TERT mutation is related to the occurrence of PTC, along with features like age, tumor diameter, extrathyroidal extension, and advanced stages III/IV in PTC patients [32, 33]. Co-mutation of TERT and BRAF are more common and have a stronger connection with clinicopathological aggressiveness. The present study also agrees that TERT mutation is an independent factor for a poor prognosis of PTC [17, 21, 34,35,36] (Table 4). This suggests that TERT detection should be included in the pathological assessment of PTC.

RAS was first discovered in thyroid tumors by Lemoine et al. [37] in 1988, and it is an oncogene that participates in the formation and development of various tumors. Point mutation or insertion mutation in the coding region of the RAS can activate the gene, and the activated RAS can increase the expression of its product, P21 protein. P21 protein participates in regulating cell growth and differentiation, it also can promote the abnormal proliferation of normal cells and ultimately transform them into tumor cells [38, 39]. RAS mutation was detected in 18 patients with PTC, with a mutation rate of 2.11%. The results showed that RAS mutation was related to the histological subtypes and was unrelated to other clinicopathological features. KRAS, NRAS, HRAS are common in PTC, especially in the follicular subtype, but lack significant and independent prognostic effects [40]. Additionally, we statistically analyzed the NTRK and PIK3CA, and found no correlation between the genetic mutations and the clinicopathological features of PTC patients.

The NGS test report includes the genetic “mutation abundance” indicator, but previous studies have not clarified the meaning of this indicator [41, 42]. In this study, BRAF V600E and RET patients were divided into low mutation abundance and high mutation abundance groups based on a threshold of 20%. The results showed that different V600E mutation abundance was associated with tumor diameter, multifocal presentation, bilateral presentation, extrathyroidal extension, lymphatic metastasis, histological subtypes and TNM staging. Different RET mutation abundance was associated with tumor capsule, but not associated with other clinicopathological features. The relationship between mutation abundance and clinicopathological features has not been reported in previous studies, and the experimental results obtained by NGS testing PTC patients’ genetic mutations have higher clinical reference value. In future studies, we can combine mutation abundance with patient prognosis information to establish a prognostic model and obtain the mutation abundance threshold. This could potentially be an independent prognostic factor.

However, this study has certain limitations. We only included 30 genes associated with PTC in our research, rather than performing whole exome sequencing. This may lead to the failure to detect some rare genes. In the future, whole exome sequencing should be conducted on PTC patients, and the sequencing results need to be combined with clinicopathological features and prognosis information to gain a more comprehensive understanding of the genetic mutations status of PTC. Furthermore, the histological subtypes included in our study are mainly classical subtype, follicular subtype, and a mixture of classical subtype and follicular subtype. Among the 855 patients, one patient was of tall-cell subtype (combined with classical subtype, and BRAF V600E mutation was detected), and one patient was of cribriform morular subtype (combined with classical subtype, and BRAF V600E mutation was detected). Due to the small number of cases and the presence of classical subtype in both, we classified them as classical subtype for analysis. In the future, large-scale research is needed to incorporate subtype such as tall-cell subtype, diffuse sclerosing subtype, and columnar-cell subtype into the study in order to further analyze and grasp the relationship between genetic mutations and histological subtypes in patients with PTC.

Conclusion

NGS technology can comprehensively analyze the genetic mutations of PTC patients, which provides important hints for the occurrence, development, diagnosis and treatment of PTC. In addition, BRAF V600E mutation, RET fusion, TERT mutation are associated with a number of high-risk clinicopathological features. Detection of genetic mutations in PTC patients by NGS is of great significance.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

BRAF:

B-Raf proto-oncogene, serine/threonine kinase

HRAS:

Harvey Rat Sarcoma Viral Oncogene Homolog

KRAS:

Kirsten Rat Sarcoma Viral Oncogene Homolog

NGS:

Next Generation Sequencing

NRAS:

Neuroblastoma RAS Viral Oncogene Homolog

NTRK:

Neurotrophic Receptor Tyrosine Kinase

PIK3CA:

Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha

PTC:

Papillary Thyroid Carcinoma

RAS:

Rat Sarcoma

RET:

Rearranged during Transfection

TERT:

Telomerase Reverse Transcriptase

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Funding

This work was supported by Key Discipline Project of Fujian Medical University Union Hospital (Department of Pathology).

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Authors and Affiliations

  1. Department of Pathology, Fujian Medical University Union Hospital, Fuzhou, 350001, Fujian, China

    Dongliang Shi, Meihong Yao, Dan Wu, Meichen Jiang, Junkang Li, Yuhui Zheng & Yinghong Yang

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Contributions

YHY and YHZ designed the study; DLS wrote the manuscript; DLS and MHY collected samples and clinical information; JKL and DW performed the experiments and acquired data; DLS and MCJ analyzed the data and drew the picture; all authors revised and approved the manuscript.

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Correspondence to Yuhui Zheng or Yinghong Yang.

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Shi, D., Yao, M., Wu, D. et al. Detection of genetic mutations in 855 cases of papillary thyroid carcinoma by next generation sequencing and its clinicopathological features. Diagn Pathol 19, 146 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13000-024-01573-3

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Keywords

Diagnostic Pathology

ISSN: 1746-1596

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