Gary L. Clayman

Context: Papillary thyroid carcinoma (PTC) carrying the BRAF mutation has been reported to be associated with high recurrence and potentially increased mortality. PTC carrying the TERT promoter mutation has been associated with older age, recurrence, and aggressive disease. Objective: The objective of this study was to determine the association of BRAF and TERT promoter gene alterations with recurrence and survival in a high-risk population. Design: Genomic DNA was analyzed for the BRAF mutation from 256 persistent/recurrent PTC (p/rPTC; 202 new, 54 previously reported) and for the TERT promoter mutation and polymorphism (242 p/rPTC). Two-tailed Fisher exact tests or the Pearson χ² test were performed for the associations between mutations and other variables. Overall and disease-free survivals were compared by log rank tests on Kaplan-Meier plots and by Cox regression analysis. TERT promoter constructs were tested in PTC cell lines to determine their activities in these cells. Results: BRAF V600E mutation was identified in 235 of 256 (91.8%), TERT promoter mutation at −124 was detected in 77 of 242 (31.8%), and TERT promoter polymorphism at −245 was found in 113 of 242 (46.7%) p/rPTC patients. A significant difference in survival was found in p/rPTC patients with the TERT promoter mutation, which also displayed increased activity in vitro as compared to the nonmutated promoter sequence. No association was noted between the BRAF mutation or TERT promoter polymorphism and recurrence or survival. A drawback of our study could be the limited number of patients with nonmutated BRAF (21 of 256 [8.2%]). Conclusions: Mutation in the TERT promoter, but not in BRAF, was associated with decreased survival in 19 (24.7%) p/rPTC patients who died of disease and in 38 (49.4%) p/rPTC patients who died at last contact. The presence or absence of the BRAF mutation and TERT promoter polymorphism, however, was not significantly correlated with survival.

Context: Cell lines are a widely used tool in cancer research. However, despite the relatively high incidence of papillary thyroid carcinoma (PTC), there are only four PTC cell lines available for international research audience. Objective: The objective of this study was to establish and characterize new PTC cell lines that represent primary tumor biology. Surgical specimens were obtained to generate PTC cell lines. Short tandem repeat profiling was used to confirm the uniqueness of the cell lines against databases of known cell lines and mutations were assessed using Sequenom. The expression of thyroid-specific genes was examined using real-time PCR. Tumorigenicity was determined using an orthotopic thyroid xenograft tumor mouse model. Results: Six PTC cell lines (five conventional PTCs and one follicular variant of PTC) were generated and found to be unique when compared by short tandem repeat profiling against databases of all existing cell lines. The five conventional PTC cell lines carry the BRAF V600E mutation and the follicular variant of PTC cell line had an NRAS mutation. Five of the six cell lines had a mutation in the promoter of the human telomerase reverse transcriptase gene. None of the cell lines have RET/PTC rearrangements. Three cell lines were tumorigenic in the orthotopic thyroid xenograft tumor mouse model. Conclusions: These five characterized conventional PTC cell lines and the unique follicular variant of PTC cell line should be valuable reagents for thyroid cancer research. The three tumorigenic cell lines can be used for in vivo testing of targeted therapeutic and novel agents.

Context: Special populations of cells that can efficiently initiate tumor growth have been characterized, and this feature supports the cancer stem cell theory. These cancer stem cell populations have been identified with CD44 and POU5F1. Most cancer stem cells express high levels of CD44 and low levels of CD24. In thyroid lesions, cancer stem cells have been detected in anaplastic carcinoma. However, little is known about the presence of cancer stem cells in papillary thyroid carcinoma (PTC), especially in recurrent PTC. Objective and Design: PTC cells were labeled and sorted by flow cytometry to obtain two populations. Total RNA was prepared from cells with high CD44 and CD24 expressions (CD44+CD24+) and from cells with high CD44 and low CD24 expressions (CD44+CD24−). The expressions of the stem cell marker POU5F1 and several differentiated thyroid markers were measured via real-time PCR. Results: CD44+CD24− cells were present in all PTCs tested, and the percentage of these cells was higher in clinically aggressive recurrent PTC than in less aggressive primary PTCs. Higher expression of POU5F1 was found in CD44+CD24− cells compared with that of CD44+CD24+ cells. The expression of POU5F1 was higher in thyrospheroids grown in serum-free condition than in cells grown in the presence of serum from the same patient, and the tumor was initiated in mice using thyrospheroids. Conclusions: The percentage of CD44+CD24− cells varied from tumor to tumor. Our findings suggest that cancer stem cells are present in PTC.

Background: Papillary thyroid carcinoma is the most common thyroid malignancy. Most papillary thyroid carcinomas contain BRAF mutations or RET/PTC rearrangements, thus providing targets for biologic therapy. Our previous studies had suggested papillary thyroid carcinomas (PTCs) with a BRAF mutation and the RET/PTC1 rearrangement have different sensitivities to MEK1/2 inhibitors, suggesting different signaling transduction pathways were involved.Methods: Src signaling transduction pathway in PTC cells was examined using Src inhibitors (PP2, SU6656, or dasatinib) and si‐Src RNA in vitro by Western blot analysis and proliferation analysis. An orthotopic xenograft mouse model was used for the in vivo studies using dasatinib.Results: In PTC cells, Src inhibitors suppressed p‐Src and p‐FAK and inhibited cell growth. In addition, significant suppression and extension of the p‐ERK1/2 dephosphorylation were detected in RET/PTC1‐rearranged cells in combination with an MEK inhibitor (CI‐1040). The Src family kinase/ABL inhibitor, dasatinib, significantly decreased tumor volume in mice inoculated with PTC cells carrying the RET/PTC1 rearrangement. In BRAF‐mutated PTC cells, Src inhibitors effectively suppressed p‐Src expression and dasatinib significantly decreased tumor volume with twice daily treatment.Conclusion: Src inhibitors effectively inhibited the Src signaling transduction pathway in PTC cells in vitro and dasatinib suppressed tumor growth in vivo. These results suggested that Src signaling transduction pathway plays an important role in regulating growth in PTC cells. Combination of Src and MEK1/2 inhibitors extended the dephosphorylation of extracellular signal‐regulated kinase (ERK)1/2 in PTCs carrying the RET/PTC1 rearrangement suggesting that combination therapy with complementary inhibitors of other signaling transduction pathways may be needed to effectively suppress growth and induce apoptosis in these cells. © 2013 Wiley Periodicals, Inc. Head Neck 36: 375–384, 2014

Background: Ultrasound (US) of the central neck compartment (CNC) is considered of limited sensitivity for nodal spread in papillary thyroid cancer (PTC); elective neck dissection is commonly advocated even in the absence of sonographic abnormalities. We hypothesized that US is an accurate predictor for long-term disease-free survival, regardless of the use of elective central neck dissection in patients with PTC. Methods: A retrospective chart review of 331 consecutive PTC patients treated with total thyroidectomy at M.D. Anderson Cancer Center between 1996 and 2003 was performed. Information retrieved included preoperative sonographic status of the CNC, surgical treatment of the neck, demographics, cancer staging, histopathological variables and use of adjuvant treatment. The endpoints for the study were nodal recurrence and survival. Results: There were 112 males and 219 females with a median age of 44 years (range 11–87). The median follow-up time for the series was 71.5 months (range 12.7–148.7). There were 151 (45.6%) patients with a T1, 58 (17.5%) with a T2, 70 (21.1%) with a T3, and 52 (15.7%) with a T4. Preoperative sonographic abnormalities were present in the CNC in 79 (23.9%) patients. During the surveillance period, 11 (3.2%) patients recurred in the central neck, with an average time for recurrence of 22.8 months. Advanced T stage (T3/T4) and abnormal US were independent prognostic factors for recurrence in the central neck (p=0.013 and p=0.005 respectively). There were 119 (35%) patients with a sonographically negative central compartment who underwent elective central neck dissection; 85 of them (71.4%) were found to be histopathologically N(+) while 34 (28.6%) were pN0. There were no differences in overall survival (p=0.32), disease specific survival (DSS; p=0.49), and recurrence-free survival (p=0.32) between these two groups. Preoperative US of the CNC was an age-independent predictor for overall survival (pp=0.0097), and disease-free survival (p=0.0005) on bivariate Cox regression. Conclusions: US of the central compartment is an age-independent predictor for survival and CNC recurrence-free survival in PTC. Prophylactic neck dissection of the central compartment does not improve long-term disease control, regardless of the histopathological status of the lymph nodes retrieved. Our findings emphasize the ability of US to clinically detect relevant nodal disease and support conservative management of the CNC in the absence of abnormal findings.

Background: Persistent or recurrent papillary thyroid carcinoma (PTC) occurs in some patients after initial thyroid surgery and often, radioactive iodine treatment. Here, we identify the efficacy, safety, and long-term outcome of our current surgical management paradigm for persistent/recurrent PTC in the central compartment in an interdisciplinary thyroid cancer clinical and research program at a tertiary thyroid cancer referral center. Methods: We retrospectively analyzed our standardized approach of comprehensive bilateral level VI/VII lymph node dissection (SND [VI, VII]) for cytologically confirmed PTC in the central compartment. Results: From 1994 to 2004, 210 patients, median age 42 (range 12–82) underwent SND (VI, VII). Most patients (106, 51%) had already undergone ≥2 surgical procedures for persistent or recurrent disease, and 31 (15%) had distant metastases at presentation. Postoperatively, 104 (71%) of the 146 patients who were thyroglobulin (Tg) positive had no evidence of disease. Anti-Tg antibodies were present in 38 patients (18%), 17 of whom (53%) did not have anti-Tg antibodies postoperatively. Fourteen patients (7%) were hypoparathyroid at presentation, and 2 more (1%) became permanently hypoparathyroid after surgery. Four patients (2%) experienced recurrent laryngeal nerve paralysis (RLNP) of a previously functioning nerve. Unanticipated RLNP was observed in only one nerve at risk. External beam radiation was given to 33 patients (17%). An additional 17 patients (8%) developed distant metastases during follow-up. At the last follow-up, 130 (66%) of the 196 patients had no detectable Tg; of these, 99 (76%) had no further evidence of disease. A median of 7.25 years after surgery, 167 (90%) of the 185 patients were without evidence of central disease, and 18 (10%) had developed central compartment recurrences within a median interval of 24.3 months. Of those with recurrence, 16 out of 18 patients (89%) underwent a subsequent surgical procedure, thus resulting in an overall 98% central compartment control rate. Kaplan–Meier disease-specific survival at 10 years was 98.9% for patients pConclusions: Bilateral comprehensive level VI/VII dissections are safe and effective for long-term control of recurrent/persistent PTC in the central lymphatic compartment.

The incidence of thyroid cancer in the United States has increased more rapidly than any other malignant disease over the past 20 years. This increase in incidence is likely influenced by improvements in diagnostic methods such as routine high-resolution ultrasonography (US) and fine-needle aspiration biopsy.¹ In recent years, preoperative staging with high-resolution US has become a standard practice in most centers. This is reflected in the 2009 American Thyroid Association Guidelines Taskforce recommendation favoring routine preoperative imaging with US over other imaging modalities for patients with thyroid cancer.²