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Exposure to ionizing radiation during childhood markedly increases the risk of developing papillary thyroid cancer. We examined tissues from 26 Ukrainian patients with thyroid cancer who were younger than 10 years of age and living in contaminated areas during the time of the Chernobyl nuclear reactor accident. We identified nonoverlapping somatic driver mutations in all 26 cases through candidate gene assays and next-generation RNA sequencing. We found that 22 tumors harbored fusion oncogenes that arose primarily through intrachromosomal rearrangements. Altogether, 23 of the oncogenic drivers identified in this cohort aberrantly activate MAPK signaling, including the 2 somatic rearrangements resulting in fusion of transcription factor ETS variant 6 (ETV6) with neurotrophic tyrosine kinase receptor, type 3 (NTRK3) and fusion of acylglycerol kinase (AGK) with BRAF. Two other tumors harbored distinct fusions leading to overexpression of the nuclear receptor PPARγ. Fusion oncogenes were less prevalent in tumors from a cohort of children with pediatric thyroid cancers that had not been exposed to radiation but were from the same geographical regions. Radiation-induced thyroid cancers provide a paradigm of tumorigenesis driven by fusion oncogenes that activate MAPK signaling or, less frequently, a PPARγ-driven transcriptional program.

Background: TERT encodes the reverse transcriptase component of telomerase, which adds telomere repeats to chromosome ends, thus enabling cell replication. Telomerase activity is required for cell immortalization. Somatic TERT promoter mutations modifying key transcriptional response elements were recently reported in several cancers, such as melanomas and gliomas., Objectives: The objectives of the study were: 1) to determine the prevalence of TERT promoter mutations C228T and C250T in different thyroid cancer histological types and cell lines; and 2) to establish the possible association of TERT mutations with mutations of BRAF, RAS, or RET/PTC., Methods: TERT promoter was PCR-amplified and sequenced in 42 thyroid cancer cell lines and 183 tumors: 80 papillary thyroid cancers (PTCs), 58 poorly differentiated thyroid cancers (PDTCs), 20 anaplastic thyroid cancers (ATCs), and 25 Hurthle cell cancers (HCCs)., Results: TERT promoter mutations were found in 98 of 225 (44%) specimens. TERT promoters C228T and C250T were mutually exclusive. Mutations were present in 18 of 80 PTCs (22.5%), in 40 of 78 (51%) advanced thyroid cancers (ATC + PDTC) (P = 3 × 10(-4) vs PTC), and in widely invasive HCCs (4 of 17), but not in minimally invasive HCCs (0 of 8). TERT promoter mutations were seen more frequently in advanced cancers with BRAF/RAS mutations compared to those that were BRAF/RAS wild-type (ATC + PDTC, 67.3 vs 24.1%; P < 10(-4)), whereas BRAF-mutant PTCs were less likely to have TERT promoter mutations than BRAF wild-type tumors (11.8 vs 50.0%; P = .04)., Conclusions: TERT promoter mutations are highly prevalent in advanced thyroid cancers, particularly those harboring BRAF or RAS mutations, whereas PTCs with BRAF or RAS mutations are most often TERT promoter wild type. Acquisition of a TERT promoter mutation could extend survival of BRAF- or RAS-driven clones and enable accumulation of additional genetic defects leading to disease progression.

Inhibitors of RET, a tyrosine kinase receptor encoded by a gene that is frequently mutated in medullary thyroid cancer, have emerged as promising novel therapies for the disease. Rapalogs and other mammalian target of rapamycin (mTOR) inhibitors are effective agents in patients with gastroenteropancreatic neuroendocrine tumors, which share lineage properties with medullary thyroid carcinomas. The objective of this study was to investigate the contribution of mTOR activity to RET-induced signaling and cell growth and to establish whether growth suppression is enhanced by co-targeting RET and mTOR kinase activities. Treatment of the RET mutant cell lines TT, TPC-1, and MZ-CRC-1 with AST487, a RET kinase inhibitor, suppressed growth and showed profound and sustained inhibition of mTOR signaling, which was recapitulated by siRNA-mediated RET knockdown. Inhibition of mTOR with INK128, a dual mTORC1 and mTORC2 kinase inhibitor, also resulted in marked growth suppression to levels similar to those seen with RET blockade. Moreover, combined treatment with AST487 and INK128 at low concentrations suppressed growth and induced apoptosis. These data establish mTOR as a key mediator of RET-mediated cell growth in thyroid cancer cells and provide a rationale for combinatorial treatments in thyroid cancers with oncogenic RET mutations.

Background: The mutated BRAF V600E protein has been specifically detected in papillary thyroid carcinomas (PTCs) using immunohistochemical (IHC) analysis. The clonal origin of PTCs harboring BRAF mutations has recently been called into question., Objectives: The purpose of this study was 2-fold: (1) to compare BRAF V600E IHC expression in PTCs, poorly differentiated thyroid carcinomas (PDTCs), and anaplastic thyroid carcinomas (ATCs) with DNA mutation analysis; and (2) to study the distribution of BRAF V600E IHC staining within thyroid cancer tissues., Methods: Whole sections and tissue microarrays from 31 PTCs, 38 PDTCs, and 22 ATCs were subjected to both mass spectrometry genotyping for the BRAF(T1799A) mutation as well as IHC staining for BRAF V600E protein., Results: Of the 31 PTCs, 16 (52%) showed strong (3+) IHC staining and harbored BRAF(T1799A), whereas the remaining 15 (48%) showed absent/faint (0/1+) staining, and were wild type for BRAF (BRAF-wt). Only 5 of 38 (13%) PDTCs harbored mutant BRAF, and these were the only ones with moderate (2+) or 3+ IHC staining. All 14 ATCs with a staining intensity of 3+ harbored BRAF(T1799A), whereas the 2 ATCs with 0/1+ staining were BRAF-wt. Six ATCs showed staining of 2+, 5 of which had high background staining. Of those 6 cases, BRAF(T1799A) was present only in the tumor without background. Homogeneous staining was found in 13 of 14 (93%) PTCs, 3 of 3 (100%) PDTCs, and 12 of 14 ATCs (86%)., Conclusions: First, absent/faint staining for BRAF V600E correlates perfectly with the lack of the BRAF(T1799A) mutation, whereas strong staining is highly specific for the BRAF(T1799A) mutation in PTCs, PDTCs, and ATCs. Moderate staining intensity cannot be relied on and should lead to genotypic analysis. Second, homogeneous staining occurs in the vast majority of cases, demonstrating that the BRAF(T1799A) mutation is a clonal event in thyroid cancer.

Background: Radioiodine (RAI) remains the mainstay of therapy for RAI-avid (RAIA) distant metastatic thyroid carcinoma. We previously demonstrated that RAI-refractory distant metastatic thyroid cancers commonly harbor BRAF mutations. However, the molecular profile of RAIA metastatic thyroid cancer is unknown. Here we describe the mutational profile of thyroid tumors from follicular cell-derived cancer (FCDTC) patients presenting with RAIA distant metastases. In addition, we aimed to correlate clinical outcomes of RAI therapy with clinicopathological factors and tumor mutational status., Methods: We retrospectively identified 43 patients with FCDTC who had RAI uptake in the lungs and/or bones on their initial ¹³¹I postablation scan. Primary tumors were genotyped for known mutations in thyroid cancer genes. Structural response to RAI was assessed 6-18 months after each administered RAI activity and at the end of follow-up., Results: RAS, BRAF, RET/PTC, and PIK3CA mutations were found in 42, 23, 10, and 2% of tumors, respectively, and the remaining 23% were wild type. None of these patients achieved cure after repeat RAI therapies, and most patients (54%) experienced disease progression despite repeated RAI administration. There was an increased prevalence of RAS mutations in these RAIA tumors. RAS-mutant cancers were more likely to concentrate iodine on diagnostic whole body scans. Despite this, structural response to RAI was not influenced by tumor genotype., Conclusions: RAIA metastatic FCDTC are overrepresented with RAS mutations, whereas RAI refractory metastatic thyroid cancers are enriched with BRAF mutations. Despite a seemingly preserved ability to concentrate iodine, RAI therapy is ineffective in achieving cure in most patients with RAIA metastatic FCDTC, even in RAS-mutant disease. These poor outcomes may be improved based on recent evidence that pretreatment with MAPK kinase 1/2 inhibitors enhances responses to RAI, particularly in patients with RAS-mutant tumors.

The RAF inhibitor vemurafenib (PLX4032) increases survival in patients with BRAF-mutant metastatic melanoma, but has limited efficacy in patients with colorectal cancers. Thyroid cancer cells are also comparatively refractory to RAF inhibitors. In contrast to melanomas, inhibition of mitogen-activated protein kinase (MAPK) signaling by PLX4032 is transient in thyroid and colorectal cancer cells. The rebound in extracellular signal-regulated kinase (ERK) in thyroid cells is accompanied by increased HER3 signaling caused by induction of ERBB3 (HER3) transcription through decreased promoter occupancy by the transcriptional repressors C-terminal binding protein 1 and 2 and by autocrine secretion of neuregulin-1 (NRG1). The HER kinase inhibitor lapatinib prevents MAPK rebound and sensitizes BRAF-mutant thyroid cancer cells to RAF or MAP-ERK kinase inhibitors. This provides a rationale for combining ERK pathway antagonists with inhibitors of feedback-reactivated HER signaling in this disease. The determinants of primary resistance to MAPK inhibitors vary between cancer types, due to preferential upregulation of specific receptor tyrosine kinases, and the abundance of their respective ligands.

Context: Hurthle cell cancer (HCC) is an understudied cancer with poor prognosis., Objective: Our objective was to elucidate the genomic foundations of HCC., Design and Setting: We conducted a large-scale integrated analysis of mutations, gene expression profiles, and copy number alterations in HCC at a single tertiary-care cancer institution., Methods: Mass spectrometry-based genotyping was used to interrogate hot spot point mutations in the most common thyroid oncogenes: BRAF, RET, NRAS, HRAS, KRAS, PIK3CA, MAP2K1, and AKT1. In addition, common oncogenic fusions of RET and NTRK1 as well as PAX8/PPARγ and AKAP9-BRAF were also assessed by RT-PCR. Global copy number changes and gene expression profiles were determined in the same tumor set as the mutational analyses., Results: We report that the mutational, transcriptional, and copy number profiles of HCC were distinct from those of papillary thyroid cancer and follicular thyroid cancer, indicating HCC to be a unique type of thyroid malignancy. Unsupervised hierarchical clustering of gene expression showed the 3 groups of Hurthle tumors (Hurthle cell adenoma [HA], minimally invasive Hurthle cell carcinoma [HMIN], and widely invasive Hurthle cell carcinoma [HWIDE] clustered separately with a marked difference between HWIDE and HA. Global copy number analysis also indicated distinct subgroups of tumors that may arise as HWIDE and HMIN. Molecular pathways that differentiate HA from HWIDE included the PIK3CA-Akt-mTOR and Wnt/β-catenin pathways, potentially providing a rationale for new targets for this type of malignancy., Conclusions: Our data provide evidence that HCC may be a unique thyroid cancer distinct from papillary and follicular thyroid cancer.

Importance: BRAF V600E is a prominent oncogene in papillary thyroid cancer (PTC), but its role in PTC-related patient mortality has not been established., Objective: To investigate the relationship between BRAF V600E mutation and PTC-related mortality., Design, Setting, and Participants: Retrospective study of 1849 patients (1411 women and 438 men) with a median age of 46 years (interquartile range, 34-58 years) and an overall median follow-up time of 33 months (interquartile range, 13-67 months) after initial treatment at 13 centers in 7 countries between 1978 and 2011., Main Outcomes and Measures: Patient deaths specifically caused by PTC., Results: Overall, mortality was 5.3% (45/845; 95% CI, 3.9%-7.1%) vs 1.1% (11/1004; 95% CI, 0.5%-2.0%) (P < .001) in BRAF V600E-positive vs mutation-negative patients. Deaths per 1000 person-years in the analysis of all PTC were 12.87 (95% CI, 9.61-17.24) vs 2.52 (95% CI, 1.40-4.55) in BRAF V600E-positive vs mutation-negative patients; the hazard ratio (HR) was 2.66 (95% CI, 1.30-5.43) after adjustment for age at diagnosis, sex, and medical center. Deaths per 1000 person-years in the analysis of the conventional variant of PTC were 11.80 (95% CI, 8.39-16.60) vs 2.25 (95% CI, 1.01-5.00) in BRAF V600E-positive vs mutation-negative patients; the adjusted HR was 3.53 (95% CI, 1.25-9.98). When lymph node metastasis, extrathyroidal invasion, and distant metastasis were also included in the model, the association of BRAF V600E with mortality for all PTC was no longer significant (HR, 1.21; 95% CI, 0.53-2.76). A higher BRAF V600E-associated patient mortality was also observed in several clinicopathological subcategories, but statistical significance was lost with adjustment for patient age, sex, and medical center. For example, in patients with lymph node metastasis, the deaths per 1000 person-years were 26.26 (95% CI, 19.18-35.94) vs 5.93 (95% CI, 2.96-11.86) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 4.43 [95% CI, 2.06-9.51]; adjusted HR, 1.46 [95% CI, 0.62-3.47]). In patients with distant tumor metastasis, deaths per 1000 person-years were 87.72 (95% CI, 62.68-122.77) vs 32.28 (95% CI, 16.14-64.55) in BRAF V600E-positive vs mutation-negative patients (unadjusted HR, 2.63 [95% CI, 1.21-5.72]; adjusted HR, 0.84 [95% CI, 0.27-2.62])., Conclusions and Relevance: In this retrospective multicenter study, the presence of the BRAF V600E mutation was significantly associated with increased cancer-related mortality among patients with PTC. Because overall mortality in PTC is low and the association was not independent of tumor features, how to use BRAF V600E to manage mortality risk in patients with PTC is unclear. These findings support further investigation of the prognostic and therapeutic implications of BRAF V600E status in PTC.

Background: Metastatic thyroid cancers that are refractory to radioiodine (iodine-131) are associated with a poor prognosis. In mouse models of thyroid cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists increase the expression of the sodium-iodide symporter and uptake of iodine. Their effects in humans are not known., Methods: We conducted a study to determine whether the MAPK kinase (MEK) 1 and MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness to radioiodine in patients with metastatic thyroid cancer. After stimulation with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography (PET) was performed before and 4 weeks after treatment with selumetinib (75 mg twice daily). If the second iodine-124 PET study indicated that a dose of iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or lesions, therapeutic radioiodine was administered while the patient was receiving selumetinib., Results: Of 24 patients screened for the study, 20 could be evaluated. The median age was 61 years (range, 44 to 77), and 11 patients were men. Nine patients had tumors with BRAF mutations, and 5 patients had tumors with mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the 20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS mutations). Eight of these 12 patients reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8 patients treated with radioiodine, 5 had confirmed partial responses and 3 had stable disease; all patients had decreases in serum thyroglobulin levels (mean reduction, 89%). No toxic effects of grade 3 or higher attributable by the investigators to selumetinib were observed. One patient received a diagnosis of myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with progression to acute leukemia., Conclusions: Selumetinib produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine; the effectiveness may be greater in patients with RAS-mutant disease. (Funded by the American Thyroid Association and others; ClinicalTrials.gov number, NCT00970359.).

Context: Medullary thyroid cancer (MTC) is a rare thyroid cancer that can occur sporadically or as part of a hereditary syndrome., Objective: To explore the genetic origin of MTC, we sequenced the protein coding exons of approximately 21,000 genes in 17 sporadic MTCs., Patients and Design: We sequenced the exomes of 17 sporadic MTCs and validated the frequency of all recurrently mutated genes and other genes of interest in an independent cohort of 40 MTCs comprised of both sporadic and hereditary MTC., Results: We discovered 305 high-confidence mutations in the 17 sporadic MTCs in the discovery phase, or approximately 17.9 somatic mutations per tumor. Mutations in RET, HRAS, and KRAS genes were identified as the principal driver mutations in MTC. All of the other additional somatic mutations, including mutations in spliceosome and DNA repair pathways, were not recurrent in additional tumors. Tumors without RET, HRAS, or KRAS mutations appeared to have significantly fewer mutations overall in protein coding exons., Conclusions: Approximately 90% of MTCs had mutually exclusive mutations in RET, HRAS, and KRAS, suggesting that RET and RAS are the predominant driver pathways in MTC. Relatively few mutations overall and no commonly recurrent driver mutations other than RET, HRAS, and KRAS were seen in the MTC exome.

Advanced human thyroid cancers are densely infiltrated with tumor-associated macrophages (TAMs) and this correlates with a poor prognosis. We used BRAF-induced papillary thyroid cancer (PTC) mouse models to examine the role of TAMs in PTC progression. Following conditional activation of BRAF(V600E) in murine thyroids there is an increased expression of the TAM chemoattractants Csf-1 and Ccl-2. This is followed by the development of PTCs that are densely infiltrated with TAMs that express Csf-1r and Ccr2. Targeting CCR2-expressing cells during BRAF-induction reduced TAM density and impaired PTC development. This strategy also induced smaller tumors, decreased proliferation and restored a thyroid follicular architecture in established PTCs. In PTCs from mice that lacked CSF-1 or that received a c-FMS/CSF-1R kinase inhibitor, TAM recruitment and PTC progression was impaired, recapitulating the effects of targeting CCR2-expressing cells. Our data demonstrate that TAMs are pro-tumorigenic in advanced PTCs and that they can be targeted pharmacologically, which may be potentially useful for patients with advanced thyroid cancers.

BRAF(V600E) drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAF(V600E) activation is Ras independent and it signals as a RAF-inhibitor-sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP, and generation of RAF-inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK pathway inhibition and antitumor activity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Although tyrosine-phosphorylated or activated STAT3 (pY-STAT3) is a well-described mediator of tumorigenesis, its role in thyroid cancer has not been investigated. We observed that 63 of 110 (57%) human primary papillary thyroid carcinoma (PTC) cases expressed nuclear pY-STAT3 in tumor cells, preferentially in association with the tumor stroma. An inverse relationship between pY-STAT3 expression with tumor size and the presence of distant metastases was observed. Using human thyroid cancer-derived cell lines [harboring rearranged during transfection (RET)/PTC, v-RAF murine sarcoma viral oncogene homolog B (BRAF), or rat sarcoma virus oncogene (RAS) alterations], we determined that IL-6/gp130/JAK signaling is responsible for STAT3 activation. STAT3 knockdown by shRNA in representative thyroid cancer cell lines that express high levels of pY-STAT3 had no effect on in vitro growth. However, xenografted short hairpin STAT3 cells generated larger tumors than control cells. Similarly, STAT3 deficiency in a murine model of BRAFV600E-induced PTC led to thyroid tumors that were more proliferative and larger than those tumors expressing STAT3wt. Genome expression analysis revealed that STAT3 knockdown resulted in the down-regulation of multiple transcripts, including the tumor suppressor insulin-like growth factor binding protein 7. Furthermore, STAT3 knockdown led to an increase in glucose consumption, lactate production, and expression of Hypoxia-inducible factor 1 (HIF1α) target genes, suggesting that STAT3 is a negative regulator of aerobic glycolysis. Our studies show that, in the context of thyroid cancer, STAT3 is paradoxically a negative regulator of tumor growth. These findings suggest that targeting STAT3 in these cancers could enhance tumor size and highlight the complexities of the role of STAT3 in tumorigenesis.

Background: Papillary thyroid carcinoma (PTC) patients presenting with cervical lymph nodes (LN) metastases (M) have a variable outcome. The objective of this study is to assess the value of meticulous histopathologic examination and genotyping in stratifying these patients into clinically relevant prognostic subgroups., Methods: This was a retrospective clinical and histopathological review of PTC patients with lymph node metastases at presentation identified between 1980 and 2002 in a single institution. Primary tumors from patients who later recurred were matched to a group of patients who did not recur and subjected to mass spectrometry genotyping encompassing the most significant oncogenes in thyroid carcinomas., Results: There were 246 patients who satisfied the inclusion criteria. The median follow-up was 10.8 years. The presence of >3 metastatic nodes was an independent predictor of decreased recurrence free survival (p=0.03). In patients <45 years, none of 45 with 1-2 metastatic LN recurred, including 26 patients followed for a median of 13 years without radioactive iodine (RAI) therapy. BRAF mutations were found in 28 (78%) of 36 genotyped tumors. Combined positivity for BRAF and extra-nodal extension was much stronger in predicting disease specific survival (DSS) (p=0.004) than the single analysis of BRAF (p=0.12) or extra-nodal extension (p=0.02)., Conclusions: (i) The number of metastatic LN is an independent predictor of recurrence in all age groups and identifies a subset of young patients with excellent prognosis who may not benefit from RAI therapy. (ii) Combined positivity for BRAF and extra-nodal extension has additive prognostic value in predicting DSS. (iii) Classification systems that assign the same magnitude of risk for recurrence or death to all patients with N1 disease should be revisited., Competing Interests: Statement The authors declare that no competing financial interests exist.

Liraglutide and exenatide are glucagon-like peptide receptor (GLP-1R) agonists used in the treatment of type 2 diabetes. Both molecules have been associated with the development of thyroid C-cell tumors after lifetime exposure in rodents. Previously, it has been reported that these tumors are preceded by increased plasma calcitonin and C-cell hyperplasia. We can now document that the murine C-cell effects are mediated via GLP-1R. Thus, 13 wk of continuous exposure to GLP-1R agonists was associated with marked increases in plasma calcitonin and in the incidence of C-cell hyperplasia in wild-type mice. In contrast, similar effects were not seen in GLP-1R knockout mice. Human C-cell cancer is often caused by activating mutations in the rearranged-during-transfection (RET) protooncogene. We developed an immunohistochemical method to assess RET activation in tissues. Liraglutide dosing to mice was not found to activate RET. Further evaluation of the signaling pathways demonstrated that liraglutide increased ribosomal S6, but not MAPK kinase, phosphorylation. These observations are consistent with effects of GLP-1R agonists on rodent C cells being mediated via mammalian target of rapamycin activation in a RET- and MAPK-independent manner.

Advanced human thyroid cancers, particularly those that are refractory to treatment with radioiodine (RAI), have a high prevalence of BRAF (v-raf murine sarcoma viral oncogene homolog B1) mutations. However, the degree to which these cancers are dependent on BRAF expression is still unclear. To address this question, we generated mice expressing one of the most commonly detected BRAF mutations in human papillary thyroid carcinomas (BRAF(V600E)) in thyroid follicular cells in a doxycycline-inducible (dox-inducible) manner. Upon dox induction of BRAF(V600E), the mice developed highly penetrant and poorly differentiated thyroid tumors. Discontinuation of dox extinguished BRAF(V600E) expression and reestablished thyroid follicular architecture and normal thyroid histology. Switching on BRAF(V600E) rapidly induced hypothyroidism and virtually abolished thyroid-specific gene expression and RAI incorporation, all of which were restored to near basal levels upon discontinuation of dox. Treatment of mice with these cancers with small molecule inhibitors of either MEK or mutant BRAF reduced their proliferative index and partially restored thyroid-specific gene expression. Strikingly, treatment with the MAPK pathway inhibitors rendered the tumor cells susceptible to a therapeutic dose of RAI. Our data show that thyroid tumors carrying BRAF(V600E) mutations are exquisitely dependent on the oncoprotein for viability and that genetic or pharmacological inhibition of its expression or activity is associated with tumor regression and restoration of RAI uptake in vivo in mice. These findings have potentially significant clinical ramifications.

Background: High-resolution ultrasound (US) is the primary tool used to identify locoregional recurrences in differentiated thyroid cancer. Although small thyroid bed (TB) nodules are a commonly reported sonographic finding, their natural history, regardless of whether they are benign or malignant, has not been well characterized. This study was designed to determine the likelihood, magnitude, and rate of growth of small TB nodules identified on routine surveillance neck US after thyroidectomy for differentiated thyroid cancer as well as to identify ultrasonographic and clinical predictors of growth., Methods: This retrospective review identified 191 patients with at least one TB nodule (≤ 11 mm) on the first postoperative US performed at a comprehensive cancer center. Change in size of each TB nodule was determined using serial US studies over time. Clinicopathologic and sonographic characteristics were analyzed as possible predictors for growth of the TB nodules., Results: Over a median clinical follow-up of 5 years, 9% (17/191) of patients had increase in size of at least one TB nodule. Median size of the TB nodules was 5 mm (range: 2-11 mm). Suspicious US features were seen in 63% (121/191) of patients with TB nodules identified on initial US and in 31% (21/67) of those with TB nodules detected on subsequent follow-up US. The rate of growth was 1.3 mm/year in those nodules showing an increase in size and thus demonstrated a significant increase in size only after several years of follow-up. The negative predictive values associated with the absence of any suspicious US features (0.97), the absence of abnormal cervical lymph nodes (0.94), and the lack of a rising serum thyroglobulin (0.93) provided clinically useful information regarding the likelihood that nodules would not increase in size., Conclusion: Most TB nodules do not show clinically significant growth over several years of follow-up. Thus, TB nodules can be followed up with cautious observation and serial ultrasonography using an approach similar to that recommended by the American Thyroid Association thyroid cancer guidelines for the management of small abnormal cervical lymph nodes.

Mice with thyroid-specific expression of oncogenic BRAF (Tg-Braf) develop papillary thyroid cancers (PTCs) that are locally invasive and have well-defined foci of poorly differentiated thyroid carcinoma (PDTC). To investigate the PTC-PDTC progression, we performed a microarray analysis using RNA from paired samples of PDTC and PTC collected from the same animals by laser capture microdissection. Analysis of eight paired samples revealed a profound deregulation of genes involved in cell adhesion and intracellular junctions, with changes consistent with an epithelial-mesenchymal transition (EMT). This was confirmed by immunohistochemistry, as vimentin expression was increased and E-cadherin lost in PDTC compared with adjacent PTC. Moreover, PDTC stained positively for phospho-Smad2, suggesting a role for transforming growth factor (TGF)β in mediating this process. Accordingly, TGFβ-induced EMT in primary cultures of thyroid cells from Tg-Braf mice, whereas wild-type thyroid cells retained their epithelial features. TGFβ-induced Smad2 phosphorylation, transcriptional activity and induction of EMT required mitogen-activated protein kinase (MAPK) pathway activation in Tg-Braf thyrocytes. Hence, tumor initiation by oncogenic BRAF renders thyroid cells susceptible to TGFβ-induced EMT, through a MAPK-dependent process.

Context: Elevated levels of TSH markedly enhance the effectiveness of radioiodine (RAI) therapy in metastatic thyroid cancer., Objective: The objective of the study was to compare short-term overall survival in thyroid cancer patients with RAI-avid distant metastases prepared for RAI therapy with either traditional thyroid hormone withdrawal (THW) or recombinant human TSH (rhTSH) stimulation., Design: This was a retrospective chart review., Setting: The study was conducted at a tertiary care comprehensive cancer center., Patients: Patients included 175 patients with RAI avid metastatic disease to lung and/or bone., Interventions: In 58 patients, all RAI treatments (remnant ablation and therapy of metastatic disease) were done with rhTSH stimulation. In 35 patients, all RAI treatments were done after THW. In 82 patients, THW was used for initial RAI treatment(s) with subsequent administered activities given after rhTSH stimulation., Main Outcome Measure: Overall survival was measured., Results: After a median follow-up of 5.5 yr, there were no significant differences in overall survival between patients prepared for RAI therapy with rhTSH alone, THW alone, or THW followed by rhTSH (Kaplan-Meier analysis, P = 0.80). In a multivariate analysis that included clinicopathological features and method of preparation (rhTSH or TWH), only age at diagnosis was an independent predictor of overall survival., Conclusions: Preparation for RAI therapy using either THW or rhTSH stimulation was associated with similar 5-yr overall survival rates in patients with RAI avid thyroid cancer metastases to lung or bone.

Mutations of BRAF are found in ∼45% of papillary thyroid cancers and are enriched in tumors with more aggressive properties. We developed mice with a thyroid-specific knock-in of oncogenic Braf (LSL-Braf(V600E)/TPO-Cre) to explore the role of endogenous expression of this oncoprotein on tumor initiation and progression. In contrast to other Braf-induced mouse models of tumorigenesis (i.e., melanomas and lung), in which knock-in of Braf(V600E) induces mostly benign lesions, Braf-expressing thyrocytes become transformed and progress to invasive carcinomas with a very short latency, a process that is dampened by treatment with an allosteric MEK inhibitor. These mice also become profoundly hypothyroid due to deregulation of genes involved in thyroid hormone biosynthesis and consequently have high TSH levels. To determine whether TSH signaling cooperates with oncogenic Braf in this process, we first crossed LSL-Braf(V600E)/TPO-Cre with TshR knockout mice. Although oncogenic Braf was appropriately activated in thyroid follicular cells of these mice, they had a lower mitotic index and were not transformed. Thyroid-specific deletion of the Gsα gene in LSL-Braf(V600E)/TPO-Cre/Gnas-E1(fl/fl) mice also resulted in an attenuated cancer phenotype, indicating that the cooperation of TshR with oncogenic Braf is mediated in part by cAMP signaling. Once tumors were established in mice with wild-type TshR, suppression of TSH did not revert the phenotype. These data demonstrate the key role of TSH signaling in Braf-induced papillary thyroid cancer initiation and provide experimental support for recent observations in humans pointing to a strong association between TSH levels and thyroid cancer incidence.

Background: A risk-adapted approach to management of thyroid cancer requires risk estimates that change over time based on response to therapy and the course of the disease. The objective of this study was to validate the American Thyroid Association (ATA) risk of recurrence staging system and determine if an assessment of response to therapy during the first 2 years of follow-up can modify these initial risk estimates., Methods: This retrospective review identified 588 adult follicular cell-derived thyroid cancer patients followed for a median of 7 years (range 1-15 years) after total thyroidectomy and radioactive iodine remnant ablation. Patients were stratified according to ATA risk categories (low, intermediate, or high) as part of initial staging. Clinical data obtained during the first 2 years of follow-up (suppressed thyroglobulin [Tg], stimulated Tg, and imaging studies) were used to re-stage each patient based on response to initial therapy (excellent, acceptable, or incomplete). Clinical outcomes predicted by initial ATA risk categories were compared with revised risk estimates obtained after response to therapy variables were used to modify the initial ATA risk estimates., Results: Persistent structural disease or recurrence was identified in 3% of the low-risk, 21% of the intermediate-risk, and 68% of the high-risk patients (p < 0.001). Re-stratification during the first 2 years of follow-up reduced the likelihood of finding persistent structural disease or recurrence to 2% in low-risk, 2% in intermediate-risk, and 14% in high-risk patients, demonstrating an excellent response to therapy (stimulated Tg < 1 ng/mL without structural evidence of disease). Conversely, an incomplete response to initial therapy (suppressed Tg > 1 ng/mL, stimulated Tg > 10 ng/mL, rising Tg values, or structural disease identification within the first 2 years of follow-up) increased the likelihood of persistent structural disease or recurrence to 13% in low-risk, 41% in intermediate-risk, and 79% in high-risk patients., Conclusions: Our data confirm that the newly proposed ATA recurrence staging system effectively predicts the risk of recurrence and persistent disease. Further, these initial ATA risk estimates can be significantly refined based on the assessment of response to initial therapy, thereby providing a dynamic risk assessment that can be used to more effectively tailor ongoing follow-up recommendations.

The follicular variant of papillary thyroid carcinoma usually presents as an encapsulated tumor and less commonly as a partially/non-encapsulated infiltrative neoplasm. The encapsulated form rarely metastasizes to lymph node, whereas infiltrative tumor often harbors nodal metastases. The molecular profile of the follicular variant was shown to be close to the follicular adenoma/carcinoma group of tumors with a high RAS and very low BRAF mutation rates. A comprehensive survey of oncogenic mutations in the follicular variant of papillary thyroid carcinoma according to its encapsulated and infiltrative forms has not been performed. Paraffin tissue from 28 patients with encapsulated and 19 with infiltrative follicular variant were subjected to mass spectrometry genotyping encompassing the most significant oncogenes in thyroid carcinomas: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1 and other related genes. There was no difference in age, gender, tumor size and angioinvasion between encapsulated or infiltrative tumors. Infiltrative carcinomas had a much higher frequency of extrathyroid extension, positive margins and nodal metastases than encapsulated tumors (P<0.05). The BRAF 1799T>A mutation was found in 5 of 19 (26%) of the infiltrative tumor and in none of the encapsulated carcinomas (P=0.007). In contrast, RAS mutations were observed in 10 of 28 (36%) of the encapsulated group (5 NRAS&#95;Q61R, 3 HRAS&#95;Q61, 1 HRAS&#95;G13C and 1 KRAS&#95;Q61R) and in only 2 of 19 (10%) of infiltrative tumors (P=0.09). One encapsulated carcinoma showed a PAX8/PPARgamma rearrangement, whereas two infiltrative tumors harbored RET/PTC fusions. Encapsulated follicular variant of papillary thyroid carcinomas have a molecular profile very close to follicular adenomas/carcinomas (high rate of RAS and absence of BRAF mutations). Infiltrative follicular variant has an opposite molecular profile closer to classical papillary thyroid carcinoma than to follicular adenoma/carcinoma (BRAF>RAS mutations). The molecular profile of encapsulated and infiltrative follicular variant parallels their biological behavior (ie, metastatic nodal and invasive patterns).

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3., ((c)2010 AACR.)

Encapsulated thyroid tumors of follicular cell origin with high-grade features (EFHG) are unusual neoplasms. In current classification schemes, they are called atypical adenomas or follicular, papillary, or poorly differentiated carcinoma. When noninvasive, EFHG create a major therapeutic/diagnostic dilemma stemming from their rarity, low-stage, high-grade appearance, and lack of long-term follow-up studies. All cases of EFHG were defined as encapsulated tumors of follicular cell origin with at least 5 mitoses per 10 high-power fields and/or tumor necrosis. Available tissues were subjected to a thyroid carcinoma platform for mass spectrometry high-throughput genotyping, which consisted of 111 known mutations in 16 different genes: BRAF, RET, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes. Twenty-five cases met the selection criteria. Tumor necrosis was present in 56.0% (n = 14). Extensive vascular invasion was identified in 24.0% (n = 6). Eight (32%) of 25 tumors were noninvasive. Twenty-two patients (88%) were free of disease (median follow up: 8.5 years). All 8 noninvasive tumor did not recur despite focal/extensive tumor necrosis in 3 cases and a median follow-up of 11.9 years. EFHG with no vascular invasion did not recur. In patients without distant metastases at presentation (n = 24), 33% (2/6) of patients with extensive angioinvasion relapsed, whereas none of 18 with absent/focal vascular invasion recurred (P = .054). Mutations were found in 10 (45%) of 22 cases tested: 8 had NRAS codon 61, 1 KRAS codon 61, and 1 had coexistent BRAF V600E and AKT1. There was a higher frequency of RAS (9/22, 41%) than BRAF mutations (1/22, 4.5%) (P = .009). Noninvasive EFHG have an indolent behavior even in the presence of extensive tumor necrosis. EFHG with absent vascular invasion have an excellent prognosis despite the frequent occurrence of tumor necrosis. NRAS mutations are the most frequent oncogenic event in EFHG., (Copyright 2010 Elsevier Inc. All rights reserved.)

Tyrosine phosphorylation plays a critical role in regulating cellular function and is a central feature in signaling cascades involved in oncogenesis. The regulation of tyrosine phosphorylation is coordinately controlled by kinases and phosphatases (PTPs). Whereas activation of tyrosine kinases has been shown to play vital roles in tumor development, the role of PTPs is much less well defined. Here, we show that the receptor protein tyrosine phosphatase delta (PTPRD) is frequently inactivated in glioblastoma multiforme (GBM), a deadly primary neoplasm of the brain. PTPRD is a target of deletion in GBM, often via focal intragenic loss. In GBM tumors that do not possess deletions in PTPRD, the gene is frequently subject to cancer-specific epigenetic silencing via promoter CpG island hypermethylation (37%). Sequencing of the PTPRD gene in GBM and other primary human tumors revealed that the gene is mutated in 6% of GBMs, 13% of head and neck squamous cell carcinomas, and in 9% of lung cancers. These mutations were deleterious. In total, PTPRD inactivation occurs in >50% of GBM tumors, and loss of expression predicts for poor prognosis in glioma patients. Wild-type PTPRD inhibits the growth of GBM and other tumor cells, an effect not observed with PTPRD alleles harboring cancer-specific mutations. Human astrocytes lacking PTPRD exhibited increased growth. PTPRD was found to dephosphorylate the oncoprotein STAT3. These results implicate PTPRD as a tumor suppressor on chromosome 9p that is involved in the development of GBMs and multiple human cancers.

Patients with poorly differentiated thyroid cancers (PDTC), anaplastic thyroid cancers (ATC), and radioactive iodine-refractory (RAIR) differentiated thyroid cancers have a high mortality, particularly if positive on [(18)F]fluorodeoxyglucose (FDG)-positron emission tomography (PET). To obtain comprehensive genetic information on advanced thyroid cancers, we designed an assay panel for mass spectrometry genotyping encompassing the most significant oncogenes in this disease: 111 mutations in RET, BRAF, NRAS, HRAS, KRAS, PIK3CA, AKT1, and other related genes were surveyed in 31 cell lines, 52 primary tumors (34 PDTC and 18 ATC), and 55 RAIR, FDG-PET-positive recurrences and metastases (nodal and distant) from 42 patients. RAS mutations were more prevalent than BRAF (44 versus 12%; P = 0.002) in primary PDTC, whereas BRAF was more common than RAS (39 versus 13%; P = 0.04) in PET-positive metastatic PDTC. BRAF mutations were highly prevalent in ATC (44%) and in metastatic tumors from RAIR PTC patients (95%). Among patients with multiple metastases, 9 of 10 showed between-sample concordance for BRAF or RAS mutations. By contrast, 5 of 6 patients were discordant for mutations of PIK3CA or AKT1. AKT1&#95;G49A was found in 9 specimens, exclusively in metastases. This is the first documentation of AKT1 mutation in thyroid cancer. Thus, RAIR, FDG-PET-positive metastases are enriched for BRAF mutations. If BRAF is mutated in the primary, it is likely that the metastases will harbor the defect. By contrast, absence of PIK3CA/AKT1 mutations in one specimen may not reflect the status at other sites because these mutations arise during progression, an important consideration for therapies directed at phosphoinositide 3-kinase effectors.

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.

We developed mice with germline endogenous expression of oncogenic Hras to study effects on development and mechanisms of tumor initiation. They had high perinatal mortality, abnormal cranial dimensions, defective dental ameloblasts, and nasal septal deviation, consistent with some of the features of human Costello syndrome. These mice developed papillomas and angiosarcomas, which were associated with Hras(G12V) allelic imbalance and augmented Hras signaling. Endogenous expression of Hras(G12V) was also associated with a higher mutation rate in vivo. Tumor initiation by Hras(G12V) likely requires augmentation of signal output, which in papillomas and angiosarcomas is achieved via increased Hras-gene copy number, which may be favored by a higher mutation frequency in cells expressing the oncoprotein.

Constitutive activation of MAPK in cancer occurs through activating mutations or overexpression of upstream effectors in the pathway, primarily of genes encoding receptor tyrosine kinases, RAS and BRAF. Arguably, the evidence for MAPK activation is most compelling in thyroid cancers and in melanomas. In this review we discuss the mechanisms of tumor development by oncogenic BRAF in these two cancer cell lineages, since this kinase signals preferentially through this pathway. We describe recent information on the mediators of BRAF-induced tumor initiation and escape from senescence. In addition, we review the biochemical events implicated in cellular growth triggered by oncogenic BRAF and the determinants of oncogene addiction. The biology of thyroid cancers induced by oncogenic BRAF is quite distinct, both in humans and in mice. There is great interest in using these insights to design rational new therapies, for which it will become crucial to understand the determinants of sensitivity and resistance to compounds designed to block the pathway. In thyroid cancer, this interest is further heightened by new information on the role of activated BRAF and MAPK pathway activation in disrupting iodine transport and thyroid hormonogenesis.

There is now a reasonably good understanding of the key oncogenic events involved in the initiation and progression of thyroid cancer. Many of these are characteristic of certain tumor types, and their presence conveys diagnostic and prognostic information. It is not yet clear how this information will be applied to clinical practice. Based on preclinical evidence, mutations of genes encoding certain kinases may also predict response to specific tyrosine kinase inhibitors, although this has not yet been explored systematically in clinical trials.

Thyroid cancers are infiltrated with tumor-associated macrophages (TAMs), yet their role in cancer progression is not known. The objectives of this study were to characterize the density of TAMs in well-differentiated (WDTC), poorly differentiated (PDTC), and anaplastic thyroid cancers (ATC) and to correlate TAM density with clinicopathologic parameters. Immunohistochemistry was performed on tissue microarray sections from WDTC (n=33), PDTC (n=37), and ATC (n=20) using macrophage-specific markers. Electronic medical records were used to gather clinical and pathologic data. Follow-up information of PDTC patients was available for 0-12 years. In total, 9 out of 33 WDTC (27%), 20 out of 37 PDTC (54%), and 19 out of 20 ATC (95%) had an increased density of CD68(+) TAMs (> or = 10 per 0.28 mm(2); WDTC versus PDTC, P=0.03; WDTC versus ATC, P<0.0001; PDTC versus ATC, P<0.002). Increased TAMs in PDTC was associated with capsular invasion (P=0.034), extrathyroidal extension (P=0.009), and decreased cancer-related survival (P=0.009) compared with PDTC with a low density of TAMs. In conclusion, the density of TAMs is increased in advanced thyroid cancers. The presence of a high density of TAMs in PDTC correlates with invasion and decreased cancer-related survival. These results suggest that TAMs may facilitate tumor progression. As novel therapies directed against thyroid tumor cell-specific targets are being tested, the potential role of TAMs as potential modulators of the thyroid cancer behavior will need to be considered.

Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated., Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis., Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines., Conclusions: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Context: Mutually exclusive mutations of RET, RAS, or BRAF are present in about 70% of papillary thyroid carcinomas, whereas only the latter two are seen in poorly differentiated and anaplastic cancers. Although the signal output common to these oncoproteins is ERK, a recent report showed that only BRAF mutations consistently predicted responsiveness to MAPK kinase (MEK) inhibitors., Objectives: Here we investigated whether sensitivity to MEK inhibition was determined by oncogene status in 13 human thyroid cancer cell lines: four with BRAF mutations, four RAS, one RET/PTC1, and four wild type., Results: Growth of BRAF (+) cells was inhibited by the MEK antagonist PD0325901 with an IC(50) of less than 5 nm. By contrast, RAS, RET/PTC1, or wild-type cells had IC(50) of 4 nm to greater than 1000 nm. Sensitivity was not predicted by coexisting mutations in PIK3CA or by PTEN status. Similar effects were obtained with the MEK inhibitor AZD6244. PD0325901 induced a sustained G1/S arrest in BRAF (+) but not BRAF (-) lines. PD0325901 was equipotent at inhibiting pERK1/2 after 2 h, regardless of genetic background, but pERK rebounded at 24 h in most lines. MEK inhibitor resistance was associated with partial refractoriness of pERK to further inhibition by the compounds. AZD6244 was more potent at inhibiting growth of NPA (BRAF +) than Cal62 (KRAS +) xenografts., Conclusion: Thyroid cancers with BRAF mutation are preferentially sensitive to MEK inhibitors, whereas tumors with other MEK-ERK effector pathway gene mutations have variable responses, either because they are only partially dependent on ERK and/or because feedback responses elicit partial refractoriness to MEK inhibition.

RET/PTC rearrangements are one of the genetic hallmarks of papillary thyroid carcinomas. RET/PTC oncoproteins lack extracellular or transmembrane domains, and activation takes place through constitutive dimerization mediated through coiled-coil motifs in the NH(2) terminus of the chimeric protein. Based on the observation that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 decreased RET/PTC kinase autophosphorylation and activation of downstream effectors in thyroid cells, despite lacking activity on the purified RET kinase, we proceeded to examine possible functional interactions between RET/PTC and EGFR. Conditional activation of RET/PTC oncoproteins in thyroid PCCL3 cells markedly induced expression and phosphorylation of EGFR, which was mediated in part through mitogen-activated protein kinase signaling. RET and EGFR were found to coimmunoprecipitate. The ability of RET to form a complex with EGFR was not dependent on recruitment of Shc or on their respective kinase activities. Ligand-induced activation of EGFR resulted in phosphorylation of a kinase-dead RET, an effect that was entirely blocked by PKI166. These effects were biologically relevant, as the EGFR kinase inhibitors PKI166, gefitinib, and AEE788 inhibited cell growth induced by various constitutively active mutants of RET in thyroid cancer cells as well as NIH3T3 cells. These data indicate that EGFR contributes to RET kinase activation, signaling, and growth stimulation and may therefore be an attractive therapeutic target in RET-induced neoplasms.

The RET kinase has emerged as a promising target for the therapy of medullary thyroid cancers (MTC) and of a subset of papillary thyroid cancers. NVP-AST487, a N,N'-diphenyl urea with an IC(50) of 0.88 mumol/L on RET kinase, inhibited RET autophosphorylation and activation of downstream effectors, and potently inhibited the growth of human thyroid cancer cell lines with activating mutations of RET but not of lines without RET mutations. NVP-AST487 induced a dose-dependent growth inhibition of xenografts of NIH3T3 cells expressing oncogenic RET, and of the MTC cell line TT in nude mice. MTCs secrete calcitonin, a useful indicator of tumor burden. Human plasma calcitonin levels derived from the TT cell xenografts were inhibited shortly after treatment, when tumor volume was still unchanged, indicating that the effects of RET kinase inhibition on calcitonin secretion were temporally dissociated from its tumor-inhibitory properties. Accordingly, NVP-AST487 inhibited calcitonin gene expression in vitro in TT cells, in part, through decreased gene transcription. These data point to a previously unknown physiologic role of RET signaling on calcitonin gene expression. Indeed, the RET ligands persephin and GDNF robustly stimulated calcitonin mRNA, which was blocked by pretreatment with NVP-AST487. Antagonists of RET kinase activity in patients with MTC may result in effects on plasma calcitonin that are either disproportionate or dissociated from the effects on tumor burden, because RET kinase mediates a physiologic pathway controlling calcitonin secretion. The role of traditional tumor biomarkers may need to be reassessed as targeted therapies designed against oncoproteins with key roles in pathogenesis are implemented.

Papillary thyroid cancers (PTC) are associated with nonoverlapping mutations of genes coding for mitogen-activated protein kinase signaling effectors (i.e., the TK receptors RET or NTRK and the signaling proteins RAS and BRAF). We examined the pattern of gene expression after activation of these oncoproteins in thyroid PCCL3 cells, with the goal of identifying pathways or gene subsets that may account for the phenotypic differences observed in human cancers. We hybridized cDNA from cells treated with or without doxycycline to induce expression of BRAF(V600E), RET/PTC3, or RET/PTC3 with small interfering RNA-mediated knockdown of BRAF, respectively, to slides arrayed with a rat 70-mer oligonucleotide library consisting of 27,342 oligos. Among the RET/PTC3-induced genes, 2,552 did not require BRAF as they were similarly regulated by RET/PTC3 with or without BRAF knockdown and not by expression of BRAF(V600E). Immune response and IFN-related genes were highly represented in this group. About 24% of RET/PTC3-regulated genes were BRAF dependent, as they were similarly modified by RET/PTC3 and BRAF(V600E) but not in cells expressing RET/PTC3 with knockdown of BRAF. A gene cluster coding for components of the mitochondrial electron transport chain pathway was down-regulated in this group, potentially altering regulation of cell viability. Metalloproteinases were also preferentially induced by BRAF, particularly matrix metalloproteinase 3 (MMP3), MMP9, and MMP13. Accordingly, conditional expression of BRAF was associated with markedly increased invasion into Matrigel compared with cells expressing RET/PTC3. The preferential induction of MMPs by BRAF could explain in part the more invasive behavior of thyroid cancers with BRAF mutations.

Purpose: Papillary thyroid carcinomas are associated with nonoverlapping activating mutations of RET, NTRK, RAS and BRAF, which altogether are present in approximately 70% of cases. We postulated that compounds that inhibit a distal effector in the mitogen-activated protein kinase (MAPK) pathway would inhibit growth and tumorigenicity of human thyroid cancer cell lines with mutations of RET or BRAF., Experimental Design and Results: We first examined the effects of AAL-881 and LBT-613, two inhibitors of RAF kinase activity, on RAF-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK activation in thyroid PCCL3 cells after conditional induction of expression of H-RAS(G12V) or BRAF(V600E). Both compounds blocked RAS and RAF-dependent MEK and ERK phosphorylation. They also potently blocked MEK phosphorylation in human thyroid cancer cell lines with either RET/PTC1 (TPC1) or BRAF(V600E) (NPA, ARO, and FRO) mutations. Inhibition of ERK phosphorylation was transient in TPC1 and ARO cells, with recovery of ERK phosphorylation associated with concomitant down-regulation of the MAPK phosphatases MKP-3 and DUSP5. Both compounds inhibited growth of all cell lines, with LBT-613 being approximately 10-fold more potent than AAL-881. TPC1 cells were more sensitive to growth inhibition (IC50 0.1-0.25 and approximately 0.05 micromol/L for AAL-881 and LBT-613, respectively) than BRAF + lines (IC50 2.5-5 and 0.1-0.5 micromol/L, respectively). Growth inhibition was associated with G1 arrest, and induction of cell death. Growth of ARO and NPA tumor xenografts was inhibited by LBT-613 or AAL-881. MEK and ERK phosphorylation was inhibited by both compounds in ARO but not in NPA cell xenografts., Conclusions: Compounds that inhibit kinase activity are effective growth inhibitors for poorly differentiated thyroid cancer cell lines with either RET or RAF mutations, and hold promise for treatment of most forms of papillary thyroid carcinoma.

Activating mutations of RAS are prevalent in thyroid follicular neoplasms, which commonly have chromosomal losses and gains. In thyroid cells, acute expression of HRAS(V12) increases the frequency of chromosomal abnormalities within one or two cell cycles, suggesting that RAS oncoproteins may interfere with cell cycle checkpoints required for maintenance of a stable genome. To explore this, PCCL3 thyroid cells with conditional expression of HRAS(V12) or HRAS(V12) effector mutants were presynchronized at the G(1)/S boundary, followed by activation of expression of RAS mutants and release from the cell cycle block. Expression of HRAS(V12) accelerated the G(2)/M phase by approximately 4 h and promoted bypass of the G(2) DNA damage and mitotic spindle checkpoints. Accelerated passage through G(2)/M and bypass of the G(2) DNA damage checkpoint, but not bypass of the mitotic spindle checkpoint, required activation of mitogen-activated protein kinase (MAPK). However, selective activation of the MAPK pathway was not sufficient to disrupt the G(2) DNA damage checkpoint, because cells arrested appropriately in G(2) despite conditional expression of HRAS(V12,S35) or BRAF(V600E). By contrast to the MAPK requirement for radiation-induced G(2) arrest, RAS-induced bypass of the mitotic spindle checkpoint was not prevented by pretreatment with MEK inhibitors. These data support a direct role for the MAPK pathway in control of G(2) progression and regulation of the G(2) DNA damage checkpoint. We propose that oncogenic RAS activation may predispose cells to genomic instability through both MAPK-dependent and independent pathways that affect critical checkpoints in G(2)/M.

In human papillary thyroid cancers (PTCs), mutations of RET/PTC, NTRK, RAS, or BRAF are found in about two thirds of cases with practically no overlap, providing genetic evidence that constitutive signaling along RET-RAS-BRAF-MAPK is key to their development. The requirement for BRAF in RET/PTC-mediated MAPK activation and gene expression has not been tested functionally. There are three RAF isoforms: ARAF, BRAF, and CRAF. Compared with the others, ARAF is a much weaker stimulator of MAPK. To determine the key RAF isoform mediating RET/PTC-induced ERK phosphorylation, we stably transfected doxycycline-inducible RET/PTC3-expressing thyroid PCCL3 cells with small interfering RNA vectors to induce selective knockdown of BRAF or CRAF. Conditional RET/PTC3 expression induced comparable ERK phosphorylation in CRAF knockdown and control cells but negligible ERK phosphorylation in BRAF knockdown cells. Selective knockdown of BRAF prevented RET/PTC-dependent down-regulation of the sodium iodide symporter, a gene that confers key biological effects of RET/PTC in PTCs. Moreover, microarray analysis revealed numerous RET/PTC-regulated genes showing requirement of BRAF for appropriate expression. These data indicate that BRAF is required for RET/PTC-induced MAPK activation in thyroid cells and support the notion that BRAF inactivation may be an attractive target for PTCs.

RET/PTC rearrangements represent key genetic events involved in papillary thyroid carcinoma (PTC) initiation. The aim of the present study was to identify the early changes in gene expression induced by RET/PTC in thyroid cells. For this purpose, microarray analysis was conducted on PCCL3 cells conditionally expressing the RET/PTC3 oncogene. Gene expression profiling 48 h after activation of RET/PTC3 identified a statistically significant modification of expression of 270 genes. Quantitative PCR confirmation of 20 of these demonstrated 90% accuracy of the microarray. Functional clustering of genes with greater than or less than 1.75-fold expression change (86 genes) revealed RET/PTC3-induced regulation of genes with key functions in apoptosis (Ripk3, Tdga), cell-cell signaling (Cdh6, Fn1), cell cycle (Il24), immune and inflammation response (Cxcl10, Scya2, Il6, Gbp2, Oas1, Tap1, RT1Aw2, C2ta, Irf1, Lmp2, Psme2, Prkr), metabolism (Aldob, Ptges, Nd2, Gss, Gstt1), signal transduction (Socs3, Nf1, Jak2, Cpg21, Dusp6, Socs1, Stat1, Stat3, Cish) and transcription (Nr4a1, Junb, Hfh1, Runx1, Foxe1). Genes coding for proteins involved in the immune response and in intracellular signal transduction pathways activated by cytokines and chemokines were strongly represented, indicating a critical role of RET/PTC3 in the early modulation of the immune response.

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.

Activating point mutations of the BRAF gene have been recently described in a variety of human tumors. In a study published in the Journal of Clinical Investigation, we reported a novel mechanism of activation of this gene via paracentric inversion of chromosome 7q. The fusion protein, AKAP9-BRAF, contains the intact kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It exhibited constitutive activation of BRAF kinase and was transforming for NIH3T3 cells. This finding represents the first demonstration of RAF activation by chromosomal rearrangement in human tumors. AKAP9-BRAF was more common in radiation-induced thyroid tumors, whereas point mutations of BRAF predominated in sporadic tumors of the same type, demonstrating the association between environmental factors and specific mechanisms of BRAF activation.

The activating mutation BRAF(T1796A) is the most prevalent genetic alteration in papillary thyroid carcinomas (PTC). It is associated with advanced PTCs, suggesting that this oncoprotein confers thyroid cancers with more aggressive properties. BRAF(T1796A) is also observed in thyroid micropapillary carcinomas and may thus be an early event in tumor development. To explore its biological consequences, we established doxycycline-inducible BRAF(V600E)-expressing clonal lines derived from well-differentiated rat thyroid PCCL3 cells. Expression of BRAF(V600E) did not induce growth in the absence of thyrotropin despite increasing DNA synthesis, which is likely explained because of a concomitant increase in apoptosis. Thyrotropin-dependent cell growth and DNA synthesis were reduced by BRAF(V600E) because of decreased thyrotropin responsiveness associated with inhibition of thyrotropin receptor gene expression. These results are similar to those obtained following conditional expression of RET/PTC. However, in contrast to RET/PTC, BRAF activation did not impair key activation steps distal to the thyrotropin receptor, such as forskolin-induced adenylyl cyclase activity or cyclic AMP-induced DNA synthesis. We reported previously that acute RET/PTC expression in PCCL3 cells did not induce genomic instability. By contrast, induction of BRAF(V600E) expression increased the frequency of micronuclei by both clastogenic and aneugenic events. These data indicate that BRAF(V600E) expression confers thyroid cells with little growth advantage because of concomitant activation of DNA synthesis and apoptosis. However, in contrast to RET/PTC, BRAF(V600E) may facilitate the acquisition of secondary genetic events through induction of genomic instability, which may account for its aggressive properties.