Objectives:
Next-generation sequencing (NGS) allows for high-throughput sequencing analysis of large regions of the human genome. We explored the use of targeted NGS for simultaneous testing for ...multiple mutations in thyroid cancer.
Design:
A custom panel (ThyroSeq) was designed to target 12 cancer genes with 284 mutational hot spots. Sequencing was performed to analyze DNA from 228 thyroid neoplastic and nonneoplastic samples including 105 frozen, 72 formalin-fixed, and 51 fine-needle aspiration samples representing all major types of thyroid cancer.
Results:
Only 5–10 ng of input DNA was sufficient for successful analysis of 99.6% of samples. The analytical accuracy for mutation detection was 100% with the sensitivity of 3%–5% of mutant allele. ThyroSeq DNA assay identified mutations in 19 of 27 of classic papillary thyroid carcinomas (PTCs) (70%), 25 of 30 follicular variant PTCs (83%), 14 of 18 conventional (78%) and 7 of 18 oncocytic follicular carcinomas (39%), 3 of 10 poorly differentiated carcinomas (30%), 20 of 27 anaplastic (ATCs) (74%), and 11 of 15 medullary thyroid carcinomas (73%). In contrast, 5 of 83 benign nodules (6%) were positive for mutations. Most tumors had a single mutation, whereas several ATCs and PTCs demonstrated two or three mutations. The most common mutations detected were BRAF and RAS followed by PIK3CA, TP53, TSHR, PTEN, GNAS, CTNNB1, and RET. The BRAF mutant allele frequency was 18%–48% in PTCs and was lower in ATCs.
Conclusions:
The ThyroSeq NGS panel allows simultaneous testing for multiple mutations with high accuracy and sensitivity, requires a small amount of DNA and can be performed in a variety of thyroid tissue and fine-needle aspiration samples, and provides quantitative assessment of mutant alleles. Using this approach, the point mutations were detected in 30%–83% of specific types of thyroid cancer and in only 6% of benign thyroid nodules and were shown to be present in the majority of cells within the cancer nodule.
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.
ObjectiveDNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary ...mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.DesignOver 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing.Results KRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).ConclusionsIn contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.
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.
Widespread clinical laboratory implementation of next-generation sequencing-based cancer testing has highlighted the importance and potential benefits of standardizing the interpretation and ...reporting of molecular results among laboratories. A multidisciplinary working group tasked to assess the current status of next-generation sequencing-based cancer testing and establish standardized consensus classification, annotation, interpretation, and reporting conventions for somatic sequence variants was convened by the Association for Molecular Pathology with liaison representation from the American College of Medical Genetics and Genomics, American Society of Clinical Oncology, and College of American Pathologists. On the basis of the results of professional surveys, literature review, and the Working Group's subject matter expert consensus, a four-tiered system to categorize somatic sequence variations based on their clinical significances is proposed: tier I, variants with strong clinical significance; tier II, variants with potential clinical significance; tier III, variants of unknown clinical significance; and tier IV, variants deemed benign or likely benign. Cancer genomics is a rapidly evolving field; therefore, the clinical significance of any variant in therapy, diagnosis, or prognosis should be reevaluated on an ongoing basis. Reporting of genomic variants should follow standard nomenclature, with testing method and limitations clearly described. Clinical recommendations should be concise and correlate with histological and clinical findings.
The MECT1/MAML2 translocation is identified in a large proportion of mucoepidermoid carcinomas (MEC) of the salivary gland and is an emerging favorable prognosticator. However, there are conflicting ...data on this translocation's specificity, restriction to low/intermediate MEC, and strength as a prognosticator. We present our experience with the MECT1/MAML2 translocation in a large cohort of MECs to address these issues. We analyzed 55 salivary MEC and 36 potential MEC mimics (24 Warthin tumors, 5 oncocytomas, 3 squamous cell carcinomas, 2 squamoid salivary duct carcinomas, 1 lymphoepithelial cyst, 1 Schneiderian carcinoma ex papilloma) for presence of the MECT1/MAML2 translocation by fluorescent in-situ hybridization (FISH) and real-time RT-PCR. Overall, MECT1/MAML2 translocation was present in 36/55 (66%) of MEC whereas all 36 non-MEC were negative for translocation. Low or intermediate-grade MEC had a higher frequency of translocation (75%) than high-grade MEC (46%) (P=0.039). Translocation positive cases had a better disease-specific survival (log rank P=0.026) although 2 patients still died of disease. Within high-grade MEC, MECT1/MAML2 positive tumors had lower rates of anaplasia (P=0.001), and mitotic counts (P=0.012). Thus, MECT1/MAML2 translocation is highly specific for MEC and imparts a better prognosis. However, it is frequent even within high-grade MEC and can be seen in lethal cases suggesting that translocation status should not supersede conventional parameters. There are 2 distinct subgroups within high-grade MEC, and the translocation negative tumors may actually be more appropriately categorized as another tumor type (such as adenosquamous carcinoma).
Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. ...Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone-independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well-differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients.
The accuracy of cancer detection in thyroid nodules by fine-needle aspiration (FNA) cytology and prognostication of thyroid cancer needs further improvement and can benefit from testing for molecular ...alterations known to occur in thyroid tumors.
Recent studies have demonstrated the feasibility of mutation detection in clinical FNA samples from thyroid nodules and their contribution to improving the diagnostic accuracy of FNA cytology. It appears that molecular testing is most beneficial for thyroid FNA samples with indeterminate cytology, where it can resolve the diagnosis in a significant number of cases. In addition to BRAF mutation, which has been studied most extensively, detection of RAS, RET/PTC, and PAX8/PPARgamma mutations also contribute substantially to cancer diagnosis. Some of these molecular markers, particularly BRAF, can also be used for tumor prognostication. In clinical setting, molecular testing of thyroid FNA samples and surgically removed tumors should utilize a restricted number of techniques that provide high accuracy and specificity of mutation detection.
Testing for cancer-specific mutations in thyroid FNA samples and surgically removed tumor tissues increases diagnostic accuracy of FNA cytology and offers better prognostication of thyroid cancer.
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