Diagnostic value and lymph node metastasis prediction of a custom‑made panel (thyroline) in thyroid cancer

Abstract

Differentiation of benign and malignant thyroid nodules is crucial for clinical management. Here, we explored the efficacy of next‑generation sequencing (NGS) in predicting the classification of benign and malignant thyroid nodules and lymph node metastasis status, and simultaneously compared the results with ultrasound (US). Thyroline was designed to detect 15 target gene mutations and 2 fusions in 98 formalin‑fixed, paraffin‑embedded (FFPE) tissues, including those from 82 thyroid cancer (TC) patients and 16 patients with benign nodules. BRAF mutations were found in 57.69% of the papillary thyroid cancer (PTC) cases, while RET mutations were detected among all the medullary thyroid cancer (MTC) cases. Multiple mutations were positive but none showed dominance in anaplastic thyroid cancer (ATC) and follicular thyroid cancer (FTC). The sensitivity and specificity of NGS prediction in differentiation of benign and malignant thyroid nodules were 79.27 and 93.75%, respectively, and the positive predictive value (PPV) and negative predictive value (NPV) were 98.48 and 46.88%, respectively. The sens-itivity and specificity of US were 76.83 and 6.25%, respectively, and the PPV and NPV were 80.77 and 5.00%, respectively. The area under curve (AUC) of NGS and US were 0.865 and 0.415, respectively. A total of 27 patients had ≥1 metastases to lymph nodes, 19 of which carried mutations, including BRAF, RET, NRAS, PIK3CA, TP53, CTNNB1 and PTEN. However, there was no correlation between the variant allele frequency of specific gene mutations and the number of metastatic lymph nodes. In conclusion, the prediction value of NGS was higher than the US‑based Thyroid Imaging Reporting and Data System (TI‑RADS). NGS is valuable for the accurate differentiation of benign and malignant thyroid nodules, and pathological subtypes in FFPE samples. The findings of the present study may pave the way for the application of NGS in analyzing fine‑needle aspiration (FNA) biopsy samples.

Figure 1.

Gene mutations and fusions in subtypes of TC and workflow of NGS. (A) BRAF, RAS, TERT, ETV6, EIF1AX, GNAS, PIK3CA, TP53 and NTRK1 mutations, as well as RET and ALK fusions, were found in PTC. BRAF, TERT, ALK fusion, GNAS, AKT1, PIK3CA, TP53 and PTEN were found in ATC. RAS, TERT, TSHR, GNAS, PENT and TP53 were found in FTC, while only RET and RAS mutations were found in MTC. (B) FFPE samples were obtained from 98 thyroid nodule patients, which was followed by CTC enumeration on NanoVelcro Chips. After collecting clinical information, we analyzed the correlation between pathological information and NGS results. (C) DNA from FFPE tissue was amplified for enrichment of target regions in a multiplex PCR reaction. Then, the library was prepared by ligating the PCR amplicons into platform-specific adapters and adding bar codes for specimen multiplexing. Finally, the library was enriched by clonal amplification (emPCR) and sequenced by massively parallel sequencing on the Ion Torrent PGM. The data analysis and variant calling were performed using bioinformatic pipelines followed by a custom SeqReporter algorithm for filtering and annotation of genetic variants. TC, thyroid cancer; NGS, next-generation sequencing; PTC, papillary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; MTC, medullary thyroid cancer; FFPE, formalin-fixed, paraffin-embedded.

Figure 2.

Gene spectrum grouped by benign and malignant thyroid nodules, pathological subtypes, metastatic lymph nodes and TI-RADS. (A) The gene spectrum of the 98 patients contained age, sex, histological diagnosis of cancer type, tumor stage, metastasis lymph node number, TI-RADS category, NGS prediction result and detailed genetic information. (B) The gene spectra of the four TC subtypes varied. For PTC, BRAF mutations were predominant, while RET mutations were detected among all the MTC. A wide range of mutations were positive but none of them showed dominance in ATC and FTC. (C) A total of 27 patients had 1 or more metastasis lymph nodes, 19 of whom carried 23 mutations, including BRAF (n=8), RET (n=8), NRAS (n=1), PIK3CA (n=2), TP53 (n=1), CTNNB1 (n=2) and PTEN (n=1). (D) Among the 78 US-predicted high-risk patients, 63 cases were diagnosed with TC by histological examination. Out of the 20 US-predicted low-risk patients, only 1 case was diagnosed with benign thyroid nodule by histological examination. TI-RADS, Thyroid Imaging Reporting and Data System; NGS, next-generation sequencing; TC, thyroid cancer; PTC, papillary thyroid cancer; MTC, medullary thyroid cancer; ATC, anaplastic thyroid cancer; FTC, follicular thyroid cancer; US, ultrasound.

Figure 3.

ROCs of NGS and US. (A) The AUC of NGS was 0.865, while the AUC of US was 0.415. (B) In situations where NGS and US drew the same conclusion, the sensitivity of combined NGS and US was 92.6% and the specificity was 50%. The AUC of combined NGS and US was 0.712. ROC, receiver operator characteristic; NGS, next-generation sequencing; US, ultrasound; AUC, area under curve.

Figure 4.

Correlation between variant allele frequency and number of metastatic lymph nodes. Spearman's analysis was performed for the variant allele frequency of BRAF, NRAS and RET mutation, RET fusion and number of metastatic lymph nodes. As a result, no correlation was found between the variant allele frequency of BRAF, NRAS and RET mutation, RET fusion and number of metastatic lymph nodes. P>0.05.

Figure 5.

Prediction difference between NGS and US for high-risk thyroid nodules. (A and B) Case 1 and 2 were diagnosed via US with benign thyroid nodule, but pathological results revealed malignancy. US showed no typical features. However, both cases were PTC with BRAF mutation. (C and D) Case 3 and 4 had consistent results among NGS, US and pathological diagnosis. US demonstrated typically malignant features, including microcalcification and hypoechogenicity. Consistently, NGS also detected TC-related driver mutations, such as NRAS and TERT in case 3, and TP53 and TERT in case 4. NGS, next-generation sequencing; US, ultrasound; PTC, papillary thyroid cancer; TC, thyroid cancer.