Yuri E. Nikiforov

2015 American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer: The American Thyroid Association Guidelines Task Force on Thyroid Nodules and Differentiated Thyroid Cancer

BACKGROUND Thyroid nodules are a common clinical problem, and differentiated thyroid cancer is becoming increasingly prevalent. Since the American Thyroid Associations (ATAs) guidelines for the management of these disorders were revised in 2009, significant scientific advances have occurred in the field. The aim of these guidelines is to inform clinicians, patients, researchers, and health policy makers on published evidence relating to the diagnosis and management of thyroid nodules and differentiated thyroid cancer. METHODS The specific clinical questions addressed in these guidelines were based on prior versions of the guidelines, stakeholder input, and input of task force members. Task force panel members were educated on knowledge synthesis methods, including electronic database searching, review and selection of relevant citations, and critical appraisal of selected studies. Published English language articles on adults were eligible for inclusion. The American College of Physicians Guideline Grading System was used for critical appraisal of evidence and grading strength of recommendations for therapeutic interventions. We developed a similarly formatted system to appraise the quality of such studies and resultant recommendations. The guideline panel had complete editorial independence from the ATA. Competing interests of guideline task force members were regularly updated, managed, and communicated to the ATA and task force members. RESULTS The revised guidelines for the management of thyroid nodules include recommendations regarding initial evaluation, clinical and ultrasound criteria for fine-needle aspiration biopsy, interpretation of fine-needle aspiration biopsy results, use of molecular markers, and management of benign thyroid nodules. Recommendations regarding the initial management of thyroid cancer include those relating to screening for thyroid cancer, staging and risk assessment, surgical management, radioiodine remnant ablation and therapy, and thyrotropin suppression therapy using levothyroxine. Recommendations related to long-term management of differentiated thyroid cancer include those related to surveillance for recurrent disease using imaging and serum thyroglobulin, thyroid hormone therapy, management of recurrent and metastatic disease, consideration for clinical trials and targeted therapy, as well as directions for future research. CONCLUSIONS We have developed evidence-based recommendations to inform clinical decision-making in the management of thyroid nodules and differentiated thyroid cancer. They represent, in our opinion, contemporary optimal care for patients with these disorders.

Molecular Testing for Mutations in Improving the Fine-Needle Aspiration Diagnosis of Thyroid Nodules

CONTEXT Thyroid nodules are common in adults, but only a small fraction of them are malignant. Fine-needle aspiration (FNA) with cytological evaluation is the most reliable tool for cancer diagnosis in thyroid nodules. However, 10-40% of nodules are diagnosed as indeterminate by cytology, making it difficult to optimally manage these patients. OBJECTIVE The aim of this study was to establish the feasibility and role of testing for tumor-specific mutations in improving the FNA diagnosis of thyroid nodules. DESIGN The prospective study included 470 FNA samples of thyroid nodules from 328 patients. At the time of aspiration, a small portion of the material was collected and tested for BRAF, RAS, RET/PTC, and PAX8/PPARgamma mutations. The mutational status was correlated with cytology and either surgical pathology diagnosis or follow-up (mean, 34 months). RESULTS A sufficient amount of nucleic acids were isolated in 98% of samples. Thirty-two mutations were found, including 18 BRAF, eight RAS, five RET/PTC, and one PAX8/PPARgamma. The presence of any mutation was a strong indicator of cancer because 31 (97%) of mutation-positive nodules had a malignant diagnosis after surgery. A combination of cytology and molecular testing showed significant improvement in the diagnostic accuracy and allowed better prediction of malignancy in the nodules with indeterminate cytology. CONCLUSIONS These results indicate that molecular testing of thyroid nodules for a panel of mutations can be effectively performed in a clinical setting. It enhances the accuracy of FNA cytology and is of particular value for thyroid nodules with indeterminate cytology.

Impact of Mutational Testing on the Diagnosis and Management of Patients with Cytologically Indeterminate Thyroid Nodules: A Prospective Analysis of 1056 FNA Samples

CONTEXT Thyroid nodules are common in adults, but only a small fraction of them is malignant. Fine-needle aspiration (FNA) cytology provides a definitive diagnosis of benign or malignant disease in many cases, whereas about 25% of nodules are indeterminate, hindering most appropriate management. OBJECTIVE The objective of the investigation was to study the clinical utility of molecular testing of thyroid FNA samples with indeterminate cytology. DESIGN Residual material from 1056 consecutive thyroid FNA samples with indeterminate cytology was used for prospective molecular analysis that included the assessment of cell adequacy by a newly developed PCR assay and testing for a panel of mutations consisted of BRAF V600E, NRAS codon 61, HRAS codon 61, and KRAS codons 12/13 point mutations and RET/PTC1, RET/PTC3, and PAX8/PPARγ rearrangements. RESULTS The collected material was adequate for molecular analysis in 967 samples (92%), which yielded 87 mutations including 19 BRAF, 62 RAS, 1 RET/PTC, and five PAX8/PPARγ. Four hundred seventy-nine patients who contributed 513 samples underwent surgery. In specific categories of indeterminate cytology, i.e. atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for a follicular neoplasm, and suspicious for malignant cells, the detection of any mutation conferred the risk of histologic malignancy of 88, 87, and 95%, respectively. The risk of cancer in mutation-negative nodules was 6, 14, and 28%, respectively. Of 6% of cancers in mutation-negative nodules with atypia of undetermined significance/follicular lesion of undetermined significance cytology, only 2.3% were invasive and 0.5% had extrathyroidal extension. CONCLUSION Molecular analysis for a panel of mutations has significant diagnostic value for all categories of indeterminate cytology and can be helpful for more effective clinical management of these patients.

MicroRNA Expression Profiling of Thyroid Tumors: Biological Significance and Diagnostic Utility

OBJECTIVE MicroRNA (miRNA) expression is deregulated in many types of human cancers. We sought to investigate the expression patterns of miRNA in all major types of thyroid tumors, including tumors carrying distinct oncogenic mutations, and to explore the utility of miRNA profiling for the preoperative diagnosis of thyroid nodules. DESIGN miRNA expression levels were detected in 60 surgically removed thyroid neoplastic and nonneoplastic samples and in 62 fine-needle aspiration (FNA) samples by RT-PCR using TaqMan MicroRNA Panel or individual miRNA sequence-specific primers. miRNA expression levels were calculated relative to normal thyroid tissue. All tumors were genotyped for most common mutations. RESULTS Various histopathological types of thyroid tumors, including those deriving from the same cell type, showed significantly different profiles of miRNA expression. Oncocytic tumors, conventional follicular tumors, papillary carcinomas, and medullary carcinomas formed distinct clusters on the unsupervised hierarchical clustering analysis. Significant correlation between miRNA expression patterns and somatic mutations was observed in papillary carcinomas. A set of seven miRNAs (miR-187, miR-221, miR-222, miR-146b, miR-155, miR-224, and miR-197) that were most differentially overexpressed in thyroid tumors vs. hyperplastic nodules in the surgical samples was validated in the FNA samples, showing high accuracy of thyroid cancer detection. CONCLUSIONS In this study, we demonstrate that various histopathological types of thyroid tumors have distinct miRNA profiles, which further differ within the same tumor type, reflecting specific oncogenic mutations. A limited set of miRNAs can be used diagnostically with high accuracy to detect thyroid cancer in the surgical and preoperative FNA samples.

Molecular genetics and diagnosis of thyroid cancer

Thyroid cancer is a common type of endocrine malignancy, and its incidence has been steadily increasing in many regions of the world. Initiation and progression of thyroid cancer involves multiple genetic and epigenetic alterations, of which mutations leading to the activation of the MAPK and PI3K–AKT signaling pathways are crucial. Common mutations found in thyroid cancer are point mutation of the BRAF and RAS genes as well as RET/PTC and PAX8/PPARγ chromosomal rearrangements. The mutational mechanisms seem to be linked to specific etiologic factors. Chromosomal rearrangements have a strong association with exposure to ionizing radiation and possibly with DNA fragility, whereas point mutations probably arise as a result of chemical mutagenesis. A potential role of dietary iodine excess in the generation of BRAF point mutations has also been proposed. Somatic mutations and other molecular alterations have been recognized as helpful diagnostic and prognostic markers for thyroid cancer and are beginning to be introduced into clinical practice, to offer a valuable tool for the management of patients with thyroid nodules.

Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas.

Papillary carcinoma is the most common type of thyroid malignancy. It has been recently shown that these tumors commonly have one of three genetic alterations: BRAF point mutations, RET/PTC rearrangements, or RAS point mutations. In this study, we analyze the relationship between these alterations and the microscopic features of papillary carcinomas, their clinical features, and prognostic characteristics. Ninety-seven papillary carcinomas were studied; in all cases, frozen tissue was available for nucleic acid extraction. Of 96 unselected cases, 42% were positive for BRAF, 18% for RET/PTC, and 15% for RAS mutations. Morphologic features were evaluated in detail in 61 cases and 6 characteristic nuclear features and 3 additional microscopic features were assessed quantitatively. At least 4 nuclear features were found in each tumor, with nuclear pseudoinclusions being the least frequent finding in all mutation groups. BRAF mutations were associated with older patient age, typical papillary appearance or the tall cell variant, a higher rate of extrathyroidal extension, and more advanced tumor stage at presentation. RET/PTC rearrangements presented at younger age and had predominantly typical papillary histology, frequent psammoma bodies, and a high rate of lymph node metastases. Tumors with RAS mutations were exclusively the follicular variant of papillary carcinoma and correlated with significantly less prominent nuclear features and low rate of lymph node metastases. These findings demonstrate that BRAF, RET/PTC, and RAS mutations are associated with distinct microscopic, clinical, and biologic features of thyroid papillary carcinomas.

Oncogenic AKAP9-BRAF fusion is a novel mechanism of MAPK pathway activation in thyroid cancer

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

RET/PTC rearrangement in thyroid tumors

Rearrangement of the RET gene, also known as RET/PTC rearrangement, is the most common genetic alteration identified to date in thyroid papillary carcinomas. The prevalence of RET/PTC in papillary carcinomas shows significant geographic variation and is approx 35% in North America. RET/PTC is more common in tumors in children and young adults, and in papillary carcinomas associated with radiation exposure. There are at least 10 different types of RET/PTC, all resulting from the fusion of the tyrosine kinase domain of RET to the 5′ portion of different genes. RET/PTC1 and RET/PTC3 are the most common types, accounting for >90% of all rearrangements. There is some evidence that different types of RET/PTC may be associated with distinct biologic properties of papillary carcinomas. RET/PTC1 tends to be more common in tumors with typical papillary growth and microcarcinomas and to have a more benign clinical course, whereas RET/PTC3 in some populations shows a strong correlation with the solid variant of papillary carcinoma and more aggressive tumor behavior. RET/PTC has recently been found in hyalinizing trabecular adenomas of the thyroid gland, providing molecular evidence in favor of this tumor to be a variant of papillary carcinoma. The occurrence of RET/PTC in Hashimoto thyroiditis and thyroid follicular adenomas and hyperplastic nodules reported in several studies has not been confirmed in other observations and remains controversial.

Molecular profile and clinical-pathologic features of the follicular variant of papillary thyroid carcinoma. An unusually high prevalence of ras mutations.

The follicular variant (FV) of papillary thyroid carcinoma is characterized by a follicular growth pattern and cytologic features of papillary carcinoma. ret/PTC rearrangements are common in classic papillary thyroid carcinoma (PTC) and PAX8-PPAR gamma and ras mutations in follicular thyroid carcinoma. Their prevalence in FV has not been established. We studied these genetic alterations and clinical-pathologic features in 30 FV cases and compared those with 46 non-FV papillary carcinomas. FV cases revealed 1 ret/PTC rearrangement (3%) and 13 ras mutations (43%). Non-FV cases harbored 13 ret/PTC (28%) (P = .006) and no ras mutations (P = .0002). No PAX8-PPAR gamma was found in either group. FV cases demonstrated a significantly higher prevalence of tumor encapsulation, angiovascular invasion, and poorly differentiated areas and a lower rate of lymph node metastases. These data indicate that the FV of papillary carcinoma has a distinct set of molecular alterations and is characterized by a high frequency of ras point mutations.

Targeted Expression of BRAFV600E in Thyroid Cells of Transgenic Mice Results in Papillary Thyroid Cancers that Undergo Dedifferentiation

The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAF2 and Tg-BRAF3) were propagated for detailed analysis. Tg-BRAF2 and Tg-BRAF3 mice had increased thyroid-stimulating hormone levels (>7- and approximately 2-fold, respectively). This likely resulted from decreased expression of thyroid peroxidase, sodium iodine symporter, and thyroglobulin. All lines seemed to successfully compensate for thyroid dysfunction, as serum thyroxine/triiodothyronine and somatic growth were normal. Thyroid glands of transgenic mice were markedly enlarged by 5 weeks of age. In Tg-BRAF2 mice, PTCs were present at 12 and 22 weeks in 14 of 15 and 13 of 14 animals, respectively, with 83% exhibiting tall-cell features, 83% areas of invasion, and 48% foci of poorly differentiated carcinoma. Tg-BRAF3 mice also developed PTCs, albeit with lower prevalence (3 of 12 and 4 of 9 at 12 and 22 weeks, respectively). Tg-BRAF2 mice had a 30% decrease in survival at 5 months. In summary, thyroid-specific expression of BRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.