Morphological changes, including nuclear score, architecture (papillary or follicular), and growth pattern (infiltrative or encapsulated), are the key points for diagnosing thyroid tumors. Based on the criteria above, major cases can be diagnosed undoubtedly. However, some cases are difficult to determine based on histological morphology alone. Compared to resected specimens, the diagnoses of biopsies are more challenging. The indeterminate rate of diagnosis is reported to be 10%-40% for FNB and 5%-20% for CNB . Our comparative study between CNB and resected specimens of thyroid nodules showed that 74 of 578 cases were unable to be determined as malignant or benign based on CNB sample morphology alone . The reason is that only follicles visible on CNB in addition to the presence of atypical nuclei and the absence of normal tissue as background makes it impossible to differentiate FTC, FVPTC, and CPTC with a follicular predominant growth pattern from FA, nodular hyperplasia and thyroiditis. Therefore, studying the application of biomarkers in distinguishing indeterminate biopsy samples is necessary.
IHC is the most popular ancillary technique used in pathological practice. Studies on resected specimens showed that CK19, Galectin-3, HBME-1 and CD56 were very helpful in discriminating malignancy from benignity [10–13]. In Dunderovic et al.’s study, the sensitivity of CK19, Galectin-3, HBME-1, and CD56 was 75.41%, 88.52%, 71.31%, and 58.20%, respectively, and the specificity of CK19, Galectin-3, HBME-1, and CD56 was 70.89%, 64.56%, 84.81%, and 92.41% . Our experience in clinical practice is similar to the results. Based on the knowledge above, it was supposed that IHC may play a role in improving the accuracy of diagnosing indeterminate biopsy samples. We searched papers published in English in PubMed and found only one focusing on this topic. In this paper, Song et al. reported that the continued indeterminate rate was 42.9% for FNB and 11.3% for CNB after IHC was applied . In our study, all 72 indeterminate samples of CNB could be determined with the help of IHC, although the accuracy of each marker was different. Taking the diagnosis of the resected specimens as the gold standard, HBME-1 and CD56 are extremely specific, with a specificity of 100% and a PPV of 100%. CK19 is the most sensitive, with a sensitivity of 93.55% and an NPV of 60.00%. Galectin-3 was less optimal, with a sensitivity of 93.55% and an NPV of 50.00%. The overexpression of HBME-1 or loss of CD56 expression strongly suggested malignancy. However, negative HBME-1 and/or positive CD56 should be cautiously considered as a sign of benignity, especially in those with overexpression of CK19 or Galectin-3 at the same time. Samples negative for HBME-1 and/or positive for CD56 as well as overexpression of CK19 or Galectin-3 should be recommended for rebiopsy, given that nodules treated with CNB are usually suspected of malignancy by ultrasound.
In the past 10 years, we have witnessed significant progress in the field of the molecular pathogenesis of thyroid carcinoma based on studies using NGS. In 2014, The Cancer Genome Atlas (TCGA) reported the comprehensive genomic characteristics of PTC. Ninety-seven percent of PTCs have unique molecular alterations, of which BRAF V600E mutations, RAS mutations, RET fusions, and TERT mutations are common, and EIF1AX mutations, ALK fusions, and NTRK1 or NTRK3 fusions are uncommon . Subsequently, the genotypes of FTC, poorly differentiated thyroid carcinoma (PDTC) and anaplastic thyroid carcinoma (ATC) have also been reported. Of the molecular alterations of FTC, RAS mutations, PAX8-PPARγ fusions and TERT mutations are common, and TSHR mutations, BRAF K601E mutations and EIF1AX mutations are uncommon. Of the molecular alterations of PDTC as well as ATC, BRAF V600E mutations, RAS mutations, TERT mutations and TP53 mutations are common [16–18]. Based on their own understanding of the mutational profile of thyroid carcinoma, researchers have tried to use diverse molecular approaches to improve the accuracy of diagnosing indeterminate biopsy samples and have presented various published results. The sensitivity and specificity of gene testing for discriminating malignancy from benignity were 63%-94% and 52%-99%, respectively, with FNB [19–21]. Regardless of how sensitive or specific it is, applying gene testing to FNB is limited in clinical practice because specialized sample collection is required at the time of the initial procedure. In contrast, CNB samples are routinely stored as paraffin-embedded blocks in which DNA can readily be extracted at any moment. In this case, gene testing is supposed to be used in distinguishing indeterminate CNB samples more practically and effectively than FNB. Compared to the numbers of studies about FNB, papers published about CNB are very limited. To date, only a few single mutations have been reported [22–25]. In this study, we detected indeterminate CNB samples by NGS using a target panel that covered the major molecular alterations of thyroid carcinoma. The sample was recorded as positive for NGS in cases of confirmed pathogenic or likely pathogenic mutations. Taking the diagnosis of the resected specimens as the gold standard, NGS is extremely specific and sensitive, with a specificity of 100%, a PPV of 100%, a sensitivity of 91.94% and an NPV of 66.67%. Considering both sensitivity and specificity, gene testing by NGS using a target panel was the most effective, with an accuracy of 93.06%. Our study shows that the application of NGS using a target panel for distinguishing indeterminate CNB samples is not only available but also very capable. In our study, all 10 cases classified as benign on resected specimens were negative for NGS on CNB, and of the 62 cases classified as malignant on resected specimens, only 5 cases were negative for NGS on CNB. Of the five, 4 cases with tumor components less than 5% were detected to have BRAF V600E mutations on resected specimens, and 1 case with tumor components of 10% was confirmed to be negative for NGS on resected specimens. Therefore, the limitation of tumor quantity is still the main reason for the weakened power of NGS in distinguishing indeterminate samples of CNB, even though the influence is lower than that of FNB. We reviewed these five cases and found that all of them were positive for CK19, Galectin-3, and HBME-1 and negative for CD56 on CNB. These results showed that IHC plays an important role in cases with negative NGS results.