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 hormoneindependent 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 ALKinduced 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 EIF1AX gene mutations have been recently found in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC). The prevalence of these mutations in other types of thyroid cancers and benign nodules is unknown. In this study, we analyzed the occurence of EIF1AX mutations in exons 2, 5 and 6 of the gene in a series of 266 thyroid tumors and hyperplastic nodules by either Sanger or next-generation sequencing (ThyroSeq v.2). In addition, 647 thyroid FNA samples with indeterminate cytology were analyzed. Using surgically removed samples, EIF1AX mutations were detected in 3/86 (2.3%) PTC, 1/4 (25%) ATC, 0/53 follicular carcinomas, 0/12 medullary carcinomas, 2/27 (7.4%) follicular adenomas and 1/80 (1.3%) hyperplastic nodules. Among 5 mutation-positive FNA samples with surgical follow-up, one nodule was PTC and others were benign follicular adenomas or hyperplastic nodules. Overall, among 33 mutations identified, A113_splice mutation at the intron 5/exon 6 splice site of EIF1AX was the most common. All 4 carcinomas harbored A113_splice mutation and three of them had one or more coexisting mutations, typically RAS. All PTC carrying EIF1AX mutation were encapsulated follicular variants. In summary, this study shows that EIF1AX mutations occur not only in thyroid carcinomas, but also in benign nodules. The most common mutation hotspot is the A113_splice, followed by a cluster of mutations in exon 2. When found in thyroid FNA samples, EIF1AX mutations confer ~20% risk of cancer; the risk is likely to be higher in nodules carrying a A113_splice mutation and when EIF1AX co-exists with RAS mutations.

Background: Hyalinizing trabecular tumor (HTT) is a rare thyroid neoplasm with a characteristic trabecular growth pattern and hyalinization. This lesion has been the subject of long-term controversy surrounding its genetic mechanisms, relationship to papillary thyroid carcinoma (PTC), and malignant potential. Due to the presence of nuclear features shared with PTC, HTT frequently contributes to a false-positive cytology, which hampers patient management. The goal of this study was to apply genome-wide sequencing analyses to elucidate the genetic mechanisms of HTT and its relationship to PTC. Methods: Whole-exome, RNA-Seq, and targeted next-generation sequencing analyses were performed to discover and characterize driver mutations in HTT. RNA-Seq results were used for pathway analysis. Tissue expression of GLIS3 and other proteins was detected by immunohistochemistry. The prevalence of GLIS fusions was studied in 17 tumors initially diagnosed as HTT, 220 PTC, and 10,165 thyroid fine-needle aspiration samples. Results: Using whole-exome and RNA-Seq analyses of the initial three HTT, no known thyroid tumor mutations were identified, while in-frame gene fusion between PAX8 exon 2 and GLIS3 exon 3 was detected in all tumors. Further analysis identified PAX8-GLIS3 in 13/14 (93%) and PAX8-GLIS1 in 1/14 (7%) of HTT confirmed after blind pathology review. The fusions were validated by Sanger sequencing and FISH. The fusions resulted in overexpression of the 3¢-portion of GLIS3 and GLIS1 mRNA containing intact DNA-binding domains of these transcription factors and upregulation of extracellular matrix genes including collagen IV. Immunohistochemistry confirmed upregulation and deposition of collagen IV and pan-collagen in HTT. The analysis of 220 PTC revealed no PAX8-GLIS3 and one PAX8-GLIS1 fusion. PAX8-GLIS3 was prospectively identified in 8/10,165 (0.1%) indeterminate cytology fineneedle aspiration samples; 5/5 resected fusion-positive nodules were HTT on surgical pathology. Conclusions: This study demonstrates that GLIS rearrangements, particularly PAX8-GLIS3, are highly prevalent in HTT but not in PTC. The fusions lead to overexpression of GLIS, upregulation of extracellular matrix genes, and deposition of collagens, which is a characteristic histopathologic feature of HTT. Due to unique genetic mechanisms and an indolent behavior, it is proposed to rename this tumor as ''GLIS-rearranged hyalinizing trabecular adenoma.''

Thyroid cancer development is driven by known point mutations or gene fusions found in ∼90% of cases, whereas driver mutations in the remaining tumors are unknown. The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays an important role in cancer, yet the mechanisms of its activation in cancer cells remain poorly understood. Using whole-transcriptome and whole-genome analyses, we identified a recurrent fusion between the thyroid adenomaassociated (THADA) gene on chromosome 2 and the LOC389473 gene on chromosome 7 located 12 kb upstream of the IGF2BP3 gene. We show that THADA fusion to LOC389473 and other regions in the vicinity does not result in the formation of a chimeric protein but instead leads to strong overexpression of the full-length IGF2BP3 mRNA and protein, increased IGF2 translation and IGF1 receptor (IGF1R) signaling via PI3K and MAPK cascades, and promotion of cell proliferation, invasion, and transformation. THADA fusions and IGF2BP3 overexpression are found in ∼5% of thyroid cancers that lack any other driver mutations. We also find that strong IGF2BP3 overexpression via gene fusion, amplification, or other mechanisms occurs in 5 to 15% of several other cancer types. Finally, we provide in vitro and in vivo evidence that growth of IGF2BP3-driven cells and tumors may be blocked by IGF1R inhibition, raising the possibility that IGF2BP3 overexpression in cancer cells may predict an anti-IGF1R benefit. thyroid cancer | genetics | chromosomal rearrangements | IGF2BP3 | IGF1R

Background Reactivation of telomerase reverse transcriptase (TERT) is an important event in cancer. Two hotspot mutations in the TERT promoter region, c.‐124C > T (C228T) and c.‐146C > T (C250T), occur in various cancer types including thyroid cancer. They generate de novo binding sites for E‐twenty‐six (ETS) transcription factors causing increased TERT transcription. The aim of this study was to search for novel TERT promoter mutations and additional mechanisms of TERT activation in thyroid cancer. Methods We studied 198 papillary thyroid carcinomas (PTCs), 34 follicular thyroid carcinomas (FTCs), 40 Hürthle cell carcinomas (HCCs), 14 poorly differentiated/anaplastic thyroid carcinomas (PDTC/ATC), and 15 medullary thyroid carcinomas (MTCs) for mutations in an −424 bp to +64 bp region of TERT. The luciferase reporter assay was used to functionally characterize the identified alterations. Copy number variations (CNVs) in the TERT region were analyzed using TaqMan copy number assay and validated with fluorescence in situ hybridization (FISH). Results We detected the hotspot c.‐124C > T and c.‐146C > T mutations in 7% PTC, 18% FTC, 25% HCC, and 86% PDTC/ATC. One PTC carried a c.‐124C > A mutation. Furthermore, we identified two novel mutations resulting in the formation of de novo ETS‐binding motifs: c.‐332C > T in one MTC and c.‐104_‐83dup in one PTC. These genetic alterations, as well as other detected mutations, led to a significant increase in TERT promoter activity when assayed using luciferase reporter system. In addition, 5% of thyroid tumors were found to have ≥3 copies of TERT. Conclusions This study confirms the increased prevalence of TERT promoter mutations and CNV in advanced thyroid cancers and describes novel functional alterations in the TERT gene promoter, including a point mutation and small duplication. These mutations, as well as TERT copy number alterations, may represent an additional mechanism of TERT activation in thyroid cancer.

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BackgroundPapillary thyroid cancer is the most common endocrine malignancy. The most sensitive and specific diagnostic tool for thyroid nodule diagnosis is fine-needle aspiration (FNA) biopsy with cytological evaluation. Nevertheless, FNA biopsy is not always decisive leading to “indeterminate” or “suspicious” diagnoses in 10 %–30 % of cases. BRAF V600E detection is currently used as molecular test to improve the diagnosis of thyroid nodules, yet it lacks sensitivity. The aim of the present study was to identify novel molecular markers/computational models to improve the discrimination between benign and malignant thyroid lesions.MethodsWe collected 118 pre-operative thyroid FNA samples. All 118 FNA samples were characterized for the presence of the BRAF V600E mutation (exon15) by pyrosequencing and further assessed for mRNA expression of four genes (KIT, TC1, miR-222, miR-146b) by quantitative polymerase chain reaction. Computational models (Bayesian Neural Network Classifier, discriminant analysis) were built, and their ability to discriminate benign and malignant tumors were tested. Receiver operating characteristic (ROC) analysis was performed and principal component analysis was used for visualization purposes.ResultsIn total, 36/70 malignant samples carried the V600E mutation, while all 48 benign samples were wild type for BRAF exon15. The Bayesian neural network (BNN) and discriminant analysis, including the mRNA expression of the four genes (KIT, TC1, miR-222, miR-146b) showed a very strong predictive value (94.12 % and 92.16 %, respectively) in discriminating malignant from benign patients. The discriminant analysis showed a correct classification of 100 % of the samples in the malignant group, and 95 % by BNN. KIT and miR-146b showed the highest diagnostic accuracy of the ROC curve, with area under the curve values of 0.973 for KIT and 0.931 for miR-146b.ConclusionsThe four genes model proposed in this study proved to be highly discriminative of the malignant status compared with BRAF assessment alone. Its implementation in clinical practice can help in identifying malignant/benign nodules that would otherwise remain suspicious.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1917-2) contains supplementary material, which is available to authorized users.

ALK fusions are found in various tumors, including thyroid cancer, and serve as a diagnostic marker and therapeutic target. Spectrum and outcomes of ALK fusions found in thyroid nodules and cancer are not fully characterized. We report a series of 44 ALK-translocated thyroid neoplasms, including 31 identified preoperatively in thyroid fine-needle aspirates (FNA). The average patients’ age was 43 years (range, 8–76 years); only one with radiation history. All 19 resected thyroid nodules with ALK fusion identified preoperatively were malignant. Among nodules with known surgical pathology (n = 32), 84% were papillary thyroid carcinomas (PTCs) and 16% poorly differentiated thyroid carcinomas (PDTCs). PTCs showed infiltrative growth with follicular architecture seen exclusively (30%) or in combination with papillary and/or solid growth (37%). Tumor multifocality was seen in 10 (31%) PTC cases. Most PDTC had a well-differentiated PTC component. Lymph node metastases were identified in 10/18 (56%) patients with neck dissection. The most common ALK fusion partners were STRN (n = 22) and EML4 (n = 17). In five cases, novel ALK fusion partners were discovered. All five PDTCs carried STRN-ALK fusion. On follow-up, ten patients were free of disease at 2–108 months, whereas two patients with PDTC died of disease. In summary, ALK fusion-positive thyroid carcinomas are typically infiltrative PTC with common follicular growth, which may show tumor dedifferentiation associated with increased mortality. Compared to EML4-ALK, STRN-ALK may be more common in PDTC, and ~10% of ALK fusions occur to rare gene partners. When ALK fusion is detected preoperatively in FNA samples, malignancy should be expected.