Short Call Abstracts

DISTINCT CYTOKINE SIGNATURES IN ANTI‐PD‐1 OR ANTI‐CLTA‐4 INDUCED THYROIDITIS: INSIGHTS FROM A NEW MOUSE MODEL

S. Ippolito^1,2, G. Di Dalmazi³, P. Chalan¹, F. Pani¹, E. Sabini¹, P. Caturegli¹

¹Pathology, Johns Hopkins University, Baltimore, MD; ²Endocrinology, Insubria University, Varese, Italy; ³Endocrinology, G. D'Annunzio University, Chieti‐Pescara, Italy

The pathogenesis of thyroiditis caused by immune checkpoint inhibitors (ICIs), such as anti‐PD‐1 and anti‐CTLA‐4, is incompletely understood. To gain mechanistic insights, we developed a mouse model of ICI‐related thyroiditis and assessed clinical, hormonal, and cytokine profiles. Forty‐three NOD‐H2^h4 mice (24 M, 19 F), 112 days old at the time of the first i.p. injection, were divided into four experimental groups: 20 mice received combined anti‐PD‐1 and anti‐CLTA‐4; 8 only anti‐PD‐1; 8 only anti‐CTLA‐4; and 7 an isotype control. Mice were sacrificed on day 172 (2 months after the initial injection) to collect thyroid gland (for histopathology and flow cytometry) and spleen (for flow cytometry). Mice were also studied before sacrifice to determine thyroid area and structure (by ultrasound using a MS700 transducer), thyroid function (by serum total T4 and TSH using bead‐based Luminex technology), and the serum levels of numerous cytokines/chemokines (by Luminex).Thyroiditis was more severe in mice receiving anti‐PD‐1 than anti‐CTLA‐4 (p = 0.01), and associated significantly with the absolute number of CD45+ infiltrating cells (cumulative OR 1.25, 95% CI 1.1–1.4, p < 0.001). On the contrary, thyroiditis was more prevalent (100% vs 63%, p < 0.01) in the anti‐CTLA‐4 mice, which also showed a larger thyroid area (17 ± 8.2 vs 11 ± 4.2 mm², p < 0.01) than those treated with anti‐PD‐1 and controls. Interestingly, mice treated with PD‐1 that developed thyroiditis showed a striking increase in systemic IL‐6 (40 pg/mL at baseline, 268 pg/mL on day 172), an increase not seen in the anti‐CTLA‐4 group (p = 0.01). IL‐6 mirrored thyroiditis severity, with highest serum values found in greatest histopathology scores (cumulative OR 1.1, 95% CI 1.02–1.15, p = 0.009). GM‐CSF and MIP1b increased more in the anti‐CTLA‐4 group (p < 0.001 for both), whereas the other cytokines/chemokines did not differ among the groups. The study reports the first mouse model of thyroiditis induced by PD‐1 blockade and, comparing it to the anti‐CTLA‐4 model, uncovers distinctive histopathological, sonographic, hormonal, and immunological features. The study also offers biomarkers, such as serum IL‐6, that could be used in the clinical setting.

PROGNOSTIC ROLE OF PRE‐SURGICAL CIRCULATING TUMORAL DNA BY NGS IN PATIENTS AFFECTED BY MEDULLARY THYROID CARCINOMA*

R. Ciampi¹, C. Romei¹, A. Tacito¹, T. Ramone¹, A. Prete¹, D. Viola¹, V. Bottici¹, L. Agate¹, E. Molinaro¹, L. Torregrossa², C. Ugolini², F. Basolo², E. Ferretti³, R. Elisei¹

¹Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy; ²Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa, Italy; ³Department of Experimental Medicine, University of Rome “La Sapienza”, Rome, Italy

Sixty% of sporadic Medullary Thyroid Carcinomas (sMTC) show mutually exclusive somatic mutations in RET and RAS. Aim of this study was to evaluate the clinical relevance of ctDNA measurement in sMTC patients before surgery. For this purpose, we studied 22 sMTC patients with plasma collected before surgery; ctDNA was analyzed by NGS‐targeted sequencing with a custom panel including the whole RET and RAS genes. Variation allele frequency (VAF) of the driver mutation in tissue and ctDNA were compared with serum calcitonin and CEA values at the time of liquid biopsy collection. Driver mutation in ctDNA was considered present when VAF >0.4%. Twenty/22 (90.9%) tumoral tissues harbored mutations in RET (n = 16), HRAS (n = 3) and KRAS (n = 1) genes with a mean VAF of 32.6%. The other 2/22 (9.1%) cases were negative and excluded from the ctDNA analysis. Among the mutated cases, 4/22 (18.2%) showed the presence of the driver mutation in their ctDNA with a mean AF value of 1.95%. Three/4 (75%) ctDNA positive cases were mutated for RET M918T and 1/4 (25%) for KRAS G12R. When comparing the presence of the driver mutation in ctDNA and Calcitonin and CEA values, we found higher values of Calcitonin [13340ng/L] in positive than in negative [1444ng/L] (P = 0.013) cases; the same was observed for CEA [526.7ng/mL vs 83.7ng/mL] (P = 0.0033). Driver mutations are detectable in liquid biopsies from sMTC patients in by NGS. Mutations in pre surgery ctDNA are more frequently detected in more advanced tumors with higher calcitonin and CEA serum values. The diagnostic and prognostic role of ctDNA may be relevant in these cases and data are promising for future application of liquid biopsy in this type of tumor.

*The study was supported by grants: AIRC‐2018‐21790 and AIFA‐2016‐02365049

TERT‐PROMOTER MUTATION INDUCES AGGRESSION IN BRAF(V600E) IN PAPILLARY THYROID CANCER: A PROBLEM OF WIDESPREAD IMMUNE SUPPRESSION

A. Chindris¹, A. Thompson¹, Y. Ma³, M. Rivera², A. Rivas⁴, J. Kachergus³, J. Casler³, X. Wang³, C. Brett⁵, V. Bernet³, R.C. Smallridge³

¹Cancer Biology, Mayo Clinic, Jacksonville, FL; ²Pathology, Mayo Clinic, Rochester, MN; ³Mayo Clinic, Jacksonville, FL; ⁴Texas Tech, Lubbock, TX; ⁵Vanderbilt, Nashville, TN

BRAF(V600E) mutation is common in papillary thyroid cancer (PTC), and most patients respond well to treatment. However, BRAF‐mutation (BRAF+) combined with a C228T and/or C250T TERT‐p mutation (TERT+) is more aggressive than BRAF+ PTC alone. PTCs frequently are infiltrated with lymphocytes. We have examined whether a TERT‐p mutation contributed to immune dysregulation in BRAF+/TERT+ vs. BRAF+TERT‐ tumors.93 PTC patients (66F; 27M; ages 20 84 yrs were segregated by mutations into BRAF+/TERT+(n = 10) or BRAF+/TERT‐(n = 83) Abundance of 797 genes was determined by multiplexed capture/detection and digital counting of mRNA transcripts using two immune function platforms (NanoString Technologies, Inc.; Seattle, WA). Tumors were reviewed by one pathologist and tumor infiltrating lymphocytes (TILs) were scored as 0; 1+(<10%); 2+(10–30%); 3+(>30%). TILs were more extensive in BRAF+/TERT‐ (TIL score >1 in 25%) than in BRAF+/TERT+(score >1 in 0%) tumors. Only 2 genes [LGALS3; PPBP (e.g.CXCL7] had a significant increase in Fold change (FC) in gene transcripts abundance in BRAF+/TERT+ vs. BRAF+/TERT‐ tumors. There were 42 genes with mRNA abundance significantly lower in BRAF+/TERT+ than in BRAF+/TERT‐ tumors, many being cytokines or chemokines.

Immune pathways were also affected. After Bonferroni correction, median pathway scores of 4 pathways (cell adhesion; lymphocyte trafficking; interferon types 1 &2) were significantly reduced in the BRAF+/TERT+ and enhanced in the BRAF+/TERT‐ tumors using the eigengene score. BRAF(V600E) mutation alters expression of many immune function genes, mostly by increasing mRNA abundance. Concomitant TERT‐p mutation often negates this effect, with most genes having reduced mRNA abundance in BRAF+/TERT+ vs. BRAF+/TERT‐ tumors. BRAF+/TERT+ tumors were larger (median = 2.2 vs. 1.5 cm), with more aggressive nodal metastases and local recurrences. BRAF+/TERT+ is associated with an immune suppressed, compared to BRAF+/TERT‐ with an immune enhanced microenvironment. This information helps explain why BRAF+/TERT+ tumors are more aggressive, and may help identify patients with advanced disease more likely to respond to immunotherapy.

THYROCYTE‐SPECIFIC ABSENCE OF MONOCARBOXYLATE TRANSPORTER (MCT) 8 DOES NOT CAUSE PAPILLARY THYROID HYPERPLASIA BUT ALTERS INTRA‐THYROIDAL THYROID HORMONE METABOLISM

K. Renko², K. Schmid³, E. Rijntjes², U. Schweizer⁴, J. Spranger¹, J. Köhrle², E.K. Wirth^1,2

¹Department of Endocrinology, Diabetes, and Nutrition, Charité‐Universitätsmedizin Berlin, Berlin, Germany; ²Institut fuer Experimentelle Endokrinologie, Charité‐Universitätsmedizin Berlin, Berlin, Germany; ³Institut für Pathologie, Universitätsklinikum Essen, Essen, Germany; ⁴Institut für Biochemie und Molekularbiologie, Rheinische Friedrich‐Wilhelms Universität Bonn, Bonn, Germany

Monocarboxylate transporter (Mct) 8 deficiency leads to papillary thyroid carcinoma (PTC) in a knockout mouse model and altered thyroid morphology in a patient carrying a non‐functional missense mutation. We therefore wanted to test whether the development of PTC depends on thyrocyte‐specific processes or is potentially mediated by systemic factors such as the increased Tsh concentration in Mct8 deficiency. We generated thyrocyte‐specific Mct8‐deficient (TPO‐Cre; Slc16a2^fl/y ) mice and performed thyroid function tests. Thyroid morphology was assessed by histology and morphometric characterization. Thyroid hormone content within the thyroid gland was evaluated by LC‐MS/MS analysis. The removal of functional Mct8 from thyrocytes leads to low circulating T₄ concentrations, while T₃and Tsh are not significantly altered. Thyrocyte‐specific Mct8‐deficient mice do not develop PTC at the age of 6 or 15 months. Morphometric analysis of thyroid glands revealed no alterations in size or distribution of follicles. However, thyrocyte‐specific Mct8 deficiency leads to lower Dio1 activity together with decreased T₃/T₄ ratios in mouse thyroid glands. The development of PTC in global Mct8 deficiency is not mediated by a specific lack of Mct8 in thyrocytes, but likely is a result of continuous stimulation through elevated Tsh. Our data support earlier conclusions that low circulating T₄ concentrations upon Mct8 deficiency are mediated by disturbed export of T₄ from the thyroid gland. Lower T₃/T₄ ratio and lower Dio1 activity in this model provokes future research to better understand intra‐thyroidal Dio1 function and regulation.

THE THYROID HORMONE RECEPTOR COACTIVATOR, MED1, REGULATES HEPATIC AUTOPHAGY AND FATTY ACID METABOLISM

J. Zhou, A. Lim, B. Singh, J. Ho, R. Sinha, P.M. Yen

Cardiovascular and Metabolic Disorders Program, Duke‐NUS Medical School, Singapore, Singapore

Thyroid hormone (TH) stimulates hepatic autophagy, b‐oxidation, oxidative phosphorylation, and mitochondrial turnover. It helps maintain normal lipid homeostasis and its action is impaired in non‐alcoholic fatty liver disease (NAFLD). Although thyroid hormone receptors (TRs) regulate autophagy, little is known about the effects of their co‐activators on autophagy and lipid metabolism. Mediator Complex Subunit 1 (MED1) is a component of the Mediator Complex that interacts with TRs to increase transcription of target genes. Accordingly, we examined MED1‘s effect on hepatic autophagy, oxidative phosphorylation, and metabolism in vtro and in vivo.siRNA knockdown (KD) of MED1 in hepatic cells decreased autophagic flux. MED1 KD also decreased oxidative phosphorylation and lipid clearance in hepatic cells pre‐loaded with free fatty acids. Furthermore, MED1 KD decreased the expression of autophagy and mitochondrial genes. T₃‐induction of autophagy, b‐oxidation, oxidative phosphorylation, and expression of mitochondrial mRNAs/proteins and classical target genes such as malic enzyme (ME1) and carnitine palmitoyltransferase 1A (CPT1A) all were reduced by MED1 KD. Additionally, Med1 KD decreased fasting‐induced autophagy, b‐oxidation, oxidative phosphorylation, and mitochondrial protein expression in hepatic cells and mouse livers. Transcriptome analyses of livers from fasted mice injected previously with MED1 or control siRNAs revealed fatty acid metabolism as a major transcriptional pathway affected by MED1 KD. The induction of serum b‐hydroxybutyrate and hepatic acylcarnitines in fasted mice was reduced by Med1 KD, and demonstrated Med1 played a key physiological role in b‐oxidation of fatty acids and ketogenesis during fasting. In summary, we have shown for the first time that a nuclear receptor co‐activator, MED1, can regulate hepatic autophagy, b‐oxidation, oxidative phosphorylation, and mitochondrial function during the basal state, and after T₃ treatment or fasting. Therefore, MED1, in addition to TRs, plays an important role in hepatic lipid metabolism during different hormonal and nutrient conditions, and its dysfunction may contribute to the over‐accumulation of hepatic triglycerides in NAFLD.

THE RISK OF ACUTE PANCREATITIS IS INCREASED IN USERS OF ANTITHYROID DRUGS, BUT DIFFERS BETWEEN METHIAMAZOL AND PROPYLTHIOURACIL. EVIDENCE FROM AF NATIONWIDE CASE‐CROSSOVER STUDY

L. Hegedus¹, T.H. Brix¹, L. lund², D. henriksen², L. folkestad¹, S. Bonnema¹, J. Hallas²

¹Endocrinology & Metabolism, Odense University hospital, Odense, Denmark; ²Clinical Pharmacology and Pharmacy, University of Southern Denmark, Odense, Denmark

The antithyroid drugs (ATD) methimazole, carbimazole (hereafter MMI for both drugs) and propylthiouracil (PTU) are used worldwide as the primary treatment for hyperthyroidism. Although serious side effects, such as agranulocytosis and hepatic dysfunction occur in 0.5–1.0%, the drugs are generally well tolerated. In January 2019, owing to six case reports on acute pancreatitis (AP) associated with MMI, The European Medicines Agency (EMA) sent out a warning, and the product labelling was changed to include AP as a serious side effect. Currently there are no data regarding PTU use and risk of AP. This prompted us to examine the association between use of the different ATD and AP in a nationwide controlled study. Registry‐based study nested within a cohort of all ever‐users (≥ one prescription) of MMI (N = 103.852) or PTU (N = 14.824) in Denmark during 1995–2018. Cases were all Danes with a hospitalization due to first‐time AP (ICD‐10; K85.x). Two approaches were used; A) a case‐crossover technique evaluating the risk of AP in ongoing MMI or PTU users. B) a case‐control study (each case with AP matched for sex and age with 4 controls) exploring a possible cumulative dose effect of MMI and PTU. Odds ratios (OR) and 95% confidence intervals (CI) for AP associated with each doubling of cumulative MMI and PTU dose, were calculated.43,580 cases with AP were identified and 226 (0.5%) and 19 (0.04%) were ongoing MMI or PTU users, respectively. The case‐crossover analysis yielded an OR of 1.51 (95% CI: 1.12–2.02) and 1.16 (0.46–2.93) for AP among ongoing MMI and PTU users, respectively. In the case‐control study there was no increased risk of AP when comparing highest vs lowest quartile of cumulative dose of ATD [ OR_MMI 0.98 (0.74–1.32) and OR_PTU 0.86 (0.37–2.04)] Doubling of cumulative MMI or PTU dose did not affect the risk of AP [OR_MMI 1.00 (0.96–1.05) and OR_PTU 1.00 (0.88–1.13)]. Ongoing MMI, but not PTU use was associated with an increased risk of acute pancreatitis. No evidence of a cumulative dose effect of MMI or PTU on the risk of AP was found. In our view, the warning by The EMA is justified since the frequency of AP in MMI users is of a similar magnitude as reported for agranulocytosis and hepatic dysfunction.

TALL CELL COMPONENT IN PAPILLARY THYROID CARCINOMA AND ITS RELATION WITH TUMOR AGGRESSIVENESS: ANALYSIS OF 3133 CONSECUTIVE CASES

A. Proietti¹, A.M. Poma², G. Russomanno¹, E. Macerola², V. Bianchini¹, F. Basolo²

¹Unit of Anatomical Pathology, University Hospital of Pisa, Pisa, PI, Italy; ²Department of Surgical, Medical, Molecular Pathology and Critical Area, University of Pisa, Pisa, PI, Italy

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer and show an increasing incidence globally. One of the most aggressive variant of PTC is tall cell variant (TCV) whose incidence varies from 1.3% to 13% of all PTC. It has been reported that TCV PTC have 10‐year overall and disease‐free survival approximately 10% shorter than that of classic PTC. In 2017 the fourth edition of the World Health Organization Classification of Tumours of Endocrine Organs defined the TCV as a PTC that contains 30% or more tall cells, however in literature there is controversy with regard to the proportion of tall cells required for the diagnosis of TCV PTC. To clarify the relation between tall cell percentage and tumor aggressiveness, 2,834 consecutive patients with 3,133 tumors diagnosed as TCV or PTC with areas of tall cells in a 17‐year period (2000–2017) were evaluated. All cases were revised according to the current classification, and were subdivided in four categories according to tall cells percentage as follows: ≤10%, 11–29%, 30–49%, ≥50%. Groups were correlated with clinicopathological parameters (age, sex, invasiveness, multifocality, node metastases, vascular invasion, presence of thyroiditis) and BRAF status, evaluated in a subgroup of 316 patients. Overall, 205 (6,5%) tumors had ≤10%, 416 (13,3%) had 11‐ 29%, 495 (15,8%) had 30–49% and 2017 (64,4%) had ≥50% tall cells. Extra‐thyroidal extension, vascular invasion and node metastases were significantly more frequent in the group 30–49% (p < 0.001); multifocality was more frequent in PTC with lower percentages of tall cells. In the group with ≥50% tall cells, tumors were significantly smaller and patients older (p < 0.0001). BRAF mutation was found in 89.9% of cases, with no statistical associations with clinical parameters. In this large series of PTC with different tall cell component, tumors having 30–49% of tall cells showed significantly higher rates of characteristics of tumors aggressiveness. This confirms that the 30% tall cell cut‐off can be considered a satisfactory value to identify neoplasms associated with higher clinical risk.

CLINICAL OUTCOMES IN A PHASE 2 TRIAL OF APATINIB IN CHINESE PATIENTS WITH PROGRESSIVE RADIOIODINE REFRACTORY DIFFERENTIATED THYROID CANCER

X. Zhang^2,3, Y. Liu^2,3, C. Wang^2,3, W. Guan⁴, J. Liang¹, Y. Lin^2,3

¹Oncology, Peking University International Hospital, Beijing, China; ²Nuclear Medicine, Peking Union Medical College (PUMC) Hospital, Chinese Academy of Medical Science & PUMC, Beijing, China; ³Beijing Key Laboratory of Molecular Targeted Diagnosis and Therapy in Nuclear Medicine, Beijing, China; ⁴Radiology, Peking Union Medical College (PUMC) Hospital, Chinese Academy of Medical Science & PUMC, Beijing, China

Patients with progressive radioiodine refractory differentiated thyroid cancer (RAIR‐DTC) usually have an unfavorable prognosis, and the treatment options are still very limited. Apatinib is an oral tyrosine kinase inhibitor (TKI) mainly targeted on the vascular endothelial growth factor receptor 2 (VEGFR‐2) having shown relatively safe and rapid responses in short‐term evaluations after 2 and 6 cycles' treatment (28 days/ cycle), but lacking a long‐term evaluation data. Twenty progressive RAIR‐DTC patients were sequentially included to receive apatinib beginning at 750 (n = 10) or 500 (n = 10) mg once daily until disease progression, unmanageable toxicity, withdrawal, or death. Study interruption and successively dose decrease (250mg/ level) due to adverse events (AEs) were permited. Efficacy and safety data, including the objective response rate (ORR), progression‐free survival (PFS) etc were evaluated during follow‐up. After a median of 13.8 months [interquartile range (IQR) 7.4, 32.3 months] of follow‐up, apatinib showed an amazing ability in tumor burden reduction with an overall ORR of 80% (90% for 750 mg schedule and 70% for 500mg schedule, P = 0.58). The overall median PFS was 18.5 months (95% confidence interval [CI], 0.0–37.4 months), which consisted of 36.8 months (95% CI 0.0–74.3 months) for 750 mg schedule and 12.9 months (95% CI 9.6–16.2 months) for 500 mg schedule without statistical significance (P > 0.05). AEs occurred in all of the patients, most of which were 1 or 2 grades. Hand‐foot skin reaction (85%), proteinuria (70%), diarrhea (65%), aspartate aminotransferase (AST) increased (65%), fatigue (60%), hypocalcemia (60%), and hypertension (55%). Within a long‐term and a small sized observation, apatinib has shown an encouraging clinical outcomes in Chinese progressive RAIR‐DTC patients.

REVOLUTIONIZING THE THERAPEUTIC LANDSCAPE OF A DEADLY DISEASE

A. Maniakas¹, R. Dadu², J.R. Wang¹, N.L. Busaidy², M. Hoffman², J. Sperling¹, N. Gross¹, E. Sturgis¹, R. Goepfert¹, S. Lai¹, M.E. Cabanillas², M.E. Zafereo¹

¹Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX; ²Endocrine Neoplasia and Hormonal Disorders, The University of Texas MD Anderson Cancer Center, Houston, TX

Anaplastic thyroid cancer (ATC) historically had median overall survival of 3–6 months from time of diagnosis with disease‐specific mortality approaching 100%. Over the last 5 years, major advancements have included accelerated access to care, better understanding of driver mutations, novel targeted therapy and immunotherapy options, and the ability to consolidate significant neoadjuvant responses with surgery and radiation. We therefore hypothesized a significant improvement in overall survival (OS) of ATC patients over the last 5 years. All patients with histopathological confirmation of ATC managed at a single tertiary care institution from January 2000 to June 2019 were included. In order to evaluated changes in OS over time, patients were divided into three groups according to date of presentation: January 2000‐December 2013, January 2014‐December 2016, January 2017‐June 2019. 2014 was the start of the Facilitated ATC Specialized Treatment team (FAST) initiative and 2017 marked the start of integrating surgery and radiation to neoadjuvant therapies. From January 2000 to June 2019, 473 patients (226F, 247M) with ATC were managed. To date, 337 (71%) deaths occurred. The median OS was 0.45 years (5.4 months), ranging from 0 to 31.9 years. Kaplan‐Meier estimates of survival at 2 years were 16.5% in the 2000–2013 group, 19.5% in the 2014–2016 group, and 36.5% in the 2017–2019 group (p < 0.001), with a hazard ratio for death of 0.756 (95% CI, 0.657–0.869) for the 2017–2019 group, as compared to a combined 2000–2016 group (p < 0.001). The era of untreatable ATC is progressively being replaced by highly specific molecular‐based personalized therapies, with the integration of other multidisciplinary therapies including surgery and radiation therapy. Landmark changes in the overall management of ATC patients over the last 5 years have led to a significant increase in their overall survival.

REDIFFERENTIATION RADIOIODINE (RAI)‐THERAPY IN PATIENTS (PTS) WITH METASTATIC PAPILLARY, FOLLICULAR AND POORLY DIFFERENTIATED THYROID CANCER (TC)

R. Falcone^1,2, S. Waguespack², R. Dadu², S.I. Sherman², N.L. Busaidy², M.I. Hu², C. Jimenez², S. Weitzman², M.E. Cabanillas²

¹Department of Translational and Precision Medicine, Sapienza University of Rome, Houston, TX; ²Endocrine Neoplasia, MD Anderson Cancer Center, Houston, TX

RAI‐refractory (RAIR) TC loses the ability to efficiently concentrate iodide, rendering RAI ineffective. Previous studies have shown improved RAI uptake after MEK inhibitors (MEKi) and/or BRAF inhibitors (BRAFi) in non‐BRAF and BRAF mutant cancers, respectively. We retrospectively evaluated 43 RAIR TC pts treated with redifferentiation (rediff) therapy using kinase inhibitor therapy (KI) with single‐agent or combination MEKi and/or BRAFi. To be included in the study pts had to have a follow up of >6 months (mos) after RAI. Our objective was to evaluate clinical response (time off KI, time to progression, progression‐free survival (PFS), response by RECIST v1.1) to rediff therapy after RAI and after stopping KI. Median age 57 (r: 34–74); 24 (56%) men; 34 (79%) pts had BRAFV600E mutation, 8 (18%) pts had RAS mutations. Among 43 pts, 15 (34%) were considered unevaluable for response to RAI as they continued KI after RAI or did not receive RAI. This left 28 pts (65%) who were treated with I131 with a median dose of 188 mCi, after which KI was discontinued. KI had been started for progressive disease in 79% of cases. 72% pts had uptake on diagnostic whole body scan (Dx WBS) and 86% on both Dx and post RAI scan. Median follow‐up was 19.5 mos after RAI. 20 (71%) pts had stable disease as best response after RAI, 4 (14%) pts had partial response; 4 pts (14%) had progression. The median time to progression was 14 mos from RAI administration and median PFS was 19.5 mos from start of KI. There were no statistical differences in PFS in BRAF and non‐BRAF mutated patients. The median time off systemic treatment was 15.5 mos. In the 28 pts, thyroglobulin (Tg) increased after KI (mean 1254 vs 646 ng/ml), decreased at 6 mos (mean 499 ng/ml) and started increasing again at the moment of progression (mean 817 ng/ml). Pts with Tg antibodies (n = 3) were excluded from Tg analysis. Rediff therapy with MEKi and/or BRAFi resensitizes some RAIR TC pts to RAI and may result in prolonged stable disease off KI. Patients who benefit from rediff therapy may avoid toxicity by discontinuing KI for a period of time. Further investigation is needed to understand which pts would likely to respond favorably to this rediff+RAI treatment strategy.

COMPLEMENT ACTIVATION IN DIFFERENT THYROID DISEASES

C. Zhao¹, Y. Yu¹, J. Liu², G. Lu¹, T. Li², Y. Gao¹, J. Zhang¹, X. Guo¹

¹Endocrinology, Peking University First Hospital, Beijing, Beijing, China; ²Pathology, Peking University First Hospital, Beijing, China

It has been demonstrated that abnormal activation of the complement system is involved in the pathogenesis of various autoimmune diseases and tumors. This study was to investigate the role of the complement system in the pathogenesis of Hashimoto's thyroiditis (HT), Graves' disease (GD), and papillary thyroid cancer (PTC). Thyroid tissues and preoperative serum were collected from patients with HT, GD, and PTC, who accepted operation in Peking University First Hospital. Normal thyroid tissues in contralateral or located outside the tumor from patients diagnosed with thyroid microscopic papillary carcinoma alone were used as control (CON) group. Deposition of C1q, mannose binding lectin (MBL), Bb, C4d, C3d and membrane attack complex (MAC) (C5b‐9), as well as the expression of CD46, CD55 and CD59, in thyroid tissues from HT, GD, PTC, and CON groups were examined by immunohistochemical staining. ELISA was used to detect serum levels of C1q, MBL, Bb, C4d, C3a, and soluble C5b‐9 (sC5b‐9) in HT, GD, PTC, and healthy donors (HD) groups. MAC could be detected in thyroid tissues of HT, GD and PTC patients, but not in normal thyroid tissues of CON group. The staining intensity of C1q showed no difference among the four groups (P > 0.05). Compared with CON group, the staining intensities of MBL, Bb, C4d, C3d and MAC in thyroid tissues of HT and PTC patients were significantly higher (all P < 0.0083). As for the complement regulatory proteins, the expression level of CD46 in thyroid tissues of HT patients was higher than that of CON group (P < 0.0083). The expression levels of CD55 and CD59 were increased in the thyroid tissues of PTC than those of HT, GD and CON groups (all P < 0.0083). The serum levels of C1q and MBL showed no significant difference among HT, GD and PTC patients and HV group (all P > 0.05). Serum levels of Bb, C4d, C3a and sC5b‐9 were significantly increased in HT, GD and PTC patients compared with HD group (all P < 0.0083). Complement overactivation in thyroid tissues of HT, GD and PTC is initiated predominantly by lectin pathway and alternative pathway. Activation of the complement system participates in the pathogenesis of HT, GD and PTC.

CD8+ AND DOUBLE NEGATIVE T LYMPHOCYTES MEDIATE DESTRUCTIVE THYROIDITIS FROM IMMUNE CHECKPOINT INHIBITORS

A. Kotwal^1,2, M. Gustafson³, S. Bornschlegl⁴, L. Kottschade⁵, D. Delivanis², A. Dietz⁴, M. Gandhi⁴, M. Ryder^2,6

¹Division of Diabetes, Endocrinology and Metabolism, University of Nebraska Medical Center, Omaha, NE; ²Division of Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic, Rochester, Rochester, MN; ³Department of Laboratory Medicine and Pathology, Mayo Clinic, Arizona, Phoenix, AZ; ⁴Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Rochester, MN; ⁵Division of Medical Oncology, Mayo Clinic, Rochester, Rochester, MN; ⁶Division of Medical Oncology, Mayo Clinic, Rochester, Rochester, MN

Immune checkpoint inhibitors (ICIs) frequently cause thyroid dysfunction but its underlying mechanism remains unclear. We have previously demonstrated the association of thyroiditis with peripheral blood circulating natural killer (NK) cells and elevated HLA‐DR surface expression on inflammatory intermediate monocytes in programmed cell death protein 1 (PD‐1) inhibitor treated patients. This study further characterizes the intra‐thyroidal immune phenotype and blood HLA haplotype associated with ICI‐induced thyroiditis. We prospectively enrolled 10 cancer patients that developed PD‐1/PD‐L1 inhibitor‐induced thyroiditis. Flow cytometry was performed on thyroid fine needle aspirates (n = 9) and peripheral blood (n = 7) compared to healthy thyroid (n = 5) and blood samples (n = 44). MHC class II haplotype was analyzed in n = 9.ICI‐induced thyroiditis patients (median age of 61 years, 50% females) demonstrated, as a % of total WBCs, less granulocytes (6.2% vs. 15.7%, p 0.0045), more mononuclear cells (MNCs) (93.8% vs. 84.3%, p 0.0045), more CD3⁺ T lymphocytes (61.3% vs. 20.1%, p 0.00006), less B lymphocytes (9.6% vs. 26.7%, p 0.0072), more double negative T lymphocytes (1.9% vs. 0.7%, p 0.006); and as a % of CD3⁺ T lymphocytes, more CD8⁺T lymphocytes (38.6% vs. 25.7%; p 0.0259) as compared to healthy thyroid samples. PD‐1 inhibitor thyroiditis patients had less CD4⁺CD28⁺ (68.1% vs. 92.2%; p 0.023) but more CD4⁺PD1⁺ (40.4% vs. 0.8%; p 0.021) and CD8⁺PD1⁺ T lymphocytes (28.8% vs. 1.5%; p 0.038) in thyroid compared to blood. Thyroiditis patients had less circulating B lymphocytes but more NK cells certain T lyphocyte subtypes (CD4⁺CD8⁺, CD4^‐CD8^‐ and gamma‐delta) and intermediate monocytes as compared to healthy volunteers. n = 6 shared HLA‐DR4 and DR53, and n = 3 shared DR15:01.ICI‐induced thyroiditis is predominantly mediated by CD8⁺ and double negative T lymphocytes, especially those that are PD1⁺. Circulating NK cells and intermediate monocytes may be a marker but don't seem to migrate to the thyroid. HLA haplotype differs from classic autoimmune thyroid disease but needs further evaluation. These findings add significantly to limited understanding of the pathophysiology behind ICI‐induced thyroiditis.

NG2 ABLATION IN MOUSE THYROID RESULTS IN THYROID DYSFUNCTION AND METABOLIC DISORDERS

F. Sui¹, X. Li¹, Q. Yang¹, H. Guan², P. Hou¹

¹ The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China; ²Endocrinology & Metabolism, Endocrinology, the First Hospital of China Medical University, Shenyang, Liaoning, China

Metabolic events including obesity, hyperlipemia and thyroid dysfunction are highly prevalent in recent years, which has been demonstrated to be closely associated with cardiovascular diseases and metabolic syndrome. Ng2 (Neuron‐glial antigen 2) also called chondroitin sulfate proteoglycan 4 (CSPG4), plays a critical role in trauma, inflammation and tumor. There is evidence showing that mice can develop obesity as a result of global ablation of the Ng2, and specific ablation of Ng2 in the central nervous system showed paradoxical phenotype. To explore the role of Ng2 in regulating thyroid function and metabolic index, we generated thyroid‐specific Ng2 knockout (Ng2 KO) mouse and Ng2‐null thyroid immortalized cells. qRT‐PCR, western blot, ELISA, IHC staining were used to test the expression of the indicated genes. A series of in vitro and in vivo studies were performed to explore the corresponding mechanism. In this study, we found that Ng2 KO mouse showed lower thyroid hormone, higher body weight, more white adipose tissue accumulation, elevated blood lipids and impaired insulin sensitivity. Our data also illustrated the possible mechanism that Ng2 depletion attenuated the activities of PI3K/AKT and MAPK/ERK signaling pathways, thus inhibited c‐Myc expression. As a core regulator of microRNA network, c‐Myc inhibited the expression of miR‐146 targeting TSHR and NG2, thereby down‐regulating their expression at post‐transcriptional levels. In summary, the present study explores the role of Ng2 in thyroid and the possible mechanism. Firstly, thyroid‐Ng2 KO mice develop thyroid dysfunctions by modulating c‐Myc/miR146/TSHR signaling axis, thereby offering a molecular basis for pathogenesis of thyroid diseases. Besides, this develop a vicious cycle (NG2‐miR146‐TSHR/NG2) for thyroid function. More importantly, Ng2 ablation in thyroid also results in various metabolic disorders which can be explained by thyroid dysfunctions, adding a novel thought to NG2 and TSHR‐related diseases, metabolic syndrome and other metabolic diseases.

A COMPETITIVE, RAPID, HOMOGENEOUS BIOASSAY FOR DETECTION OF THYROID‐BLOCKING ANTIBODIES

L.Y. Miao

R&D, Quidel Corp., Athens, OH

Recently, we developed a homogeneous bioassay (Turbo^TM TSI Bioassay) to detect thyroid stimulating antibodies. To complement this TSI bioassay, we have developed the Turbo^TM TBI Bioassay, a competitive, rapid, homogeneous bioassay that can be used to assess the activity of thyroid‐blocking antibodies (TBAb/TBI) in patient sera. The same engineered CHO K1 cell line expressing both a chimeric TSH receptor (TSHR) and a luciferase‐based homogeneous cAMP biosensor (GloSensor^TM, GS) is utilized for the detection of TBAb/TBI. In this assay, the TSHR‐blocking antibodies compete with bovine TSH (bTSH) for binding to the TSHR which influences the production of cAMP induced by the TSH‐TSHR interaction resulting in a proportional decrease in biosensor luciferase activity as compared to the bTSH reference control. The output of this assay is measured as relative light units (RLU) in a luminometer in real‐time and results of the assay are reported as percent inhibition (% I) of sample RLU compared to the reference RLU. The Turbo^TM TBI Bioassay is performed in white 96‐well plates under room temperature without wash, lysis, or pre‐incubation steps, which enables a rapid assay turnaround time of 60 minutes. Following CLSI guidelines, the Turbo^TM TBI Bioassay LoD was determined to be 33% I with a TBAb‐spiked normal serum and the preliminary assay cutoff was determined to be 40% I by testing over 140 sera containing a TSH level within the normal range. The specificity of the Turbo^TM TBI Bioassay was demonstrated by showing negative results to both a high concentration of TSAb and TSI positive serum samples, and the bioassay was unaffected by 21 potential interfering substances. The precision of the assay proved to be excellent showing an overall within laboratory precision <11% CV for all low, moderate and high TBI positive samples. When testing 16 clinical samples from patientts with Hashimoto's disease, 31% of the samples were positive for TBI by the Turbo^TMTBI Bioassay. The Turbo^TM TBI Bioassay is an important advancement in the long term effort to develop a simplified set of bioassays that can differentiate anti‐TSHR antibodies with stimulating or blocking activity.

A RAPID HOMOGENOUS BIOASSAY FOR DETECTION OF THYROID‐STIMULATING ANTIBODIES BASED ON A LUMINESCENT CYCLIC AMP BIOSENSOR

L.Y. Miao

R&D, Quidel Corp., Athens, OH

The Thyretain™ TSI Bioassay is a highly specific method to detect thyroid stimulating antibodies in the bloodstream indicative of the autoimmune disorder, Grave's Disease. To improve the performance and simplify the workflow of this bioassay and to support a semi‐quantitative result in International units of IU/L, we have generated a stable CHO‐K1 cell line expressing both a chimeric TSH receptor (TSHR) and a luciferase‐based homogeneous cAMP biosensor (GloSensor^TM, GS). A rapid, semi‐quantitative, homogenous bioassay called Turbo^TM TSI Bioassay has been developed using this cell line to measure TSI in patient sera. The assay works by measuring changes in the intracellular cAMP level induced by the binding of TSI to the TSHR. The GS luciferase reporter is then activated upon binding of cAMP and produces light that can be measured in real‐time in intact cells in a luminometer. The Turbo^TM TSI Biosssay is performed in white 96‐well plates under room temperature without wash, lysis, or pre‐incubation steps, which enables a rapid assay turnaround time of 60 minutes. Following the CLSI guideline, the LoD and LoQ of the assay were determined to be 0.018 IU/L and 0.06 IU/L, respectively. The preliminary assay cutoff was determined to be 0.024 IU/L by ROC analysis using the Thyretain^TM TSI Bioassay results as reference. The analytical sensitivity of the Turbo‐TSI bioassay was comparable to the Thyretain^TM TSI Bioassay with both assays exhibiting similar EC₅₀ values for a TSHR stimulating monoclonal antibody. The specificity of the assay was demonstrated by showing no response to a high concentration of a TSHR blocking antibody. In addition, the assay was unaffected by 21 potential interfering substances. When testing 198 clinical samples, the positive and negative percent agreement between the Turbo^TM TSI and the Thyretain^TM TSI bioassays were 98.7% and 93.5%, respectively. The Turbo^TM TSI Bioassay is user‐friendly and can be employed as a very promising advancement in the differential diagnosis of autoimmune thyroid disease (AITD).

STAG2 REGULATES GLUTAMINE METABOLISM OF THYROID CANCER CELLS THROUGH MODULATING THE ERK/AKT/GSK3β/C‐MYC FEEDBACK PATHWAY

X. Li, F. Sui, Q. Yang, B. Shi, P. Hou

The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, China

STAG2 is a crucial subunit of cohesin complex, involved in the separation of sister chromatids during cell division. Inactivation mutations of STAG2 in BRAF^V600E mutant melanoma is one of the causes of drug resistance. However, the role of STAG2 in thyroid cancer remains largely unknown.

Besides this, glutamine metabolism, which is one of the most important link in cancer metabolism, is crucial to tumor differentiation, proliferation and metastasis. Although abnormal changes of glutamine metabolism have been found in thyroid cancer, the study on which is still relatively hysteretic. Meanwhile, little is known about the role of STAG2 played in thyroid cancer glutamine metabolism. TCGA database was used to analyze STAG2 expression and mutation in thyroid cancer. Thyroid cancer cells transfected with STAG2 specific shRNA were screened and were performed with MTT, colony formation assay and drug inhibition test. Apoptosis, cell cycle assays and ROS were detected by flow cytometry. qRT‐PCR, CHX chase assay, Western blot and immunohistochemistry were done to explore the mechanism about the impact of STAG2 downregulation on thyroid cancer cells glutamine metabolism. Xenograft tumor experiment was done to evaluate therapy effect of glutaminase inhibitor in vivo especially on STAG2 downregulated thyroid cancer. Although STAG2 was down regulated in thyroid cancer, and knocking down STAG2 caused a conspicuous up‐regulation of p‐ERK, it had no remarkable influence on malignant behavior of thyroid cancer cells, or sensitivity of BRAF^V600E mutant thyroid cancer cells to MEK inhibitors. Interestingly, we discovered down‐regulated STAG2 decreased the ability to utilize glutamine in thyroid cancer cells, partly via ERK/AKT/GSK3β/c‐Myc feedback pathway, which resulted in being more dependent on glutamine supply and more sensitive to glutaminase inhibitor.STAG2 may affect glutamine metabolism of thyroid cancer cells through modulating the ERK/AKT/GSK3β/c‐Myc feedback pathway. Furthermore, thyroid cancer cells with down regulated STAG2 showed better response to glutaminase inhibitor. Thus, STAG2 partially took part in glutamine metabolism of thyroid cancer cells that it would provide a new strategy for thyroid cancer therapy.

VEGFR‐2 ANTAGONIST INHIBITS CELL PROLIFERATION, MIGRATION, INVASION VIA BLOCKING PI3K‐AKT AND MAPK SIGNALING PATHWAYS IN THYROID CANCER CELLS

C. Liu¹, Z. Liu³, P. Hou⁴, Y. Lin², X. Li¹

¹General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, Beijing, China; ²Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, Beijing, China; ³Surgical Oncology, The First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, Xi'an, Shaanxi, China; ⁴Key Laboratory for Tumor Precision Medicine of Shaanxi Province and Department of Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University College of Medicine, Xi‘an, Shaanxi, China

VEGFR‐2 mediated signaling cascades are involved in proliferation, migration, survival, and permeability changes of vascular endothelial cells. It was thought that VEGFR‐2 antagonists exerted their antitumor effects by inhibiting angiogenesis of tumor tissues. However, some recent studies have found that they have significant direct anti‐tumor effects in some tumors. The aim of this study is to explore the antitumor effects and mechanisms of VEGFR‐2 antagonist in thyroid cancer. The antitumor effects of VEGFR‐2 antagonist (Apatinib) in thyroid cancer cells were evaluated through a series of in vitro experiments, and the antitumor mechanisms of VEGFR‐2 antagonist were explored using Western‐blot. Compared with the normal human thyroid cell line HTori3, the expressions of VEGFR‐2 in thyroid cancer cell lines (including IHH4, BCPAP, TPC‐1, C643, K1, 8305C) were significantly increased, especially in C643 and 8305C cell lines. VEGFR‐2 antagonist could inhibit the proliferation of thyroid cancer cells in a dose‐dependent manner, and significantly reduce the invasion and migration of these cells, induce G0/G1 phase arrest and promote cancer cell apoptosis. Additionally, the anti‐proliferation effect of VEGFR‐2 antagonist was significantly reduced after knocking out the KDR gene. The results of Western‐blot showed that VEGFR‐2 antagonist could significantly reduce the expression of VEGFR‐2 and its phosphorylation, and further inhibit the phosphorylation of downstream molecules AKT and ERK1/2.VEGFR‐2 antagonist inhibited the cell proliferation, invasion and migration in thyroid cancer cell lines via inhibiting the PI3K‐AKT and MAPK signaling pathways, exerted direct anti‐tumor effects. Thus, directly targeting VEGFR‐2 can be an effective strategy for thyroid cancer expressing VEGFR‐2.

LNCPTC1 INDUCES CELL APOPTOSIS AND PROMOTES PROLIFERATION, MIGRATION IN PAPILLARY THYROID CANCER IN VITRO VIA INFLUENCING PHOSPHORYLATION OF KEY PROTEINS IN HIPPO PATHWAY

W. Xu, T. Liao, L. Han, B. Ma, Y. Wang

Fudan University Shanghai Cancer Center, Shanghai, China

Nowadays, thyroid cancer has been one of the most common endocrine system tumor in the female population. LncRNAs are important regulators that act by various patterns in a lot of cellular activities. In our previous study, YAP1 (Yes‐associated protein 1) can stimulate proliferation of PTC (papillary thyroid cancer), and regulate cell cycle, migration and invasion through combining with transcriptional activators, but it is still unclear that if any lncRNA is involved in this progress. We found that in 16 lncRNAs which might interact with YAP1, lncPTC1 got a higher expression level in PTC. Our study was aimed to figure out whether lncPTC1 is associated with occurrence and development in PTC and Hippo pathway. Quantitative real time polymerase chain reaction (qRT‐PCR) was performed in 70 PTC tissues and their paired normal thyroid tissues to dig out relationship among expression level of lncRNAs and several clinical pathological parameters. RNA Immunoprecipitation (RIP) and RNA pull‐down assays were applied to figure out lncRNAs that can interact with YAP1 and exact RNA combining area of YAP1. The proliferation, migration and apoptosis condition were analyzed by some cell function assays such as cell counting‐8 (CCK8), trans‐well, clone formation and flow cytometry. Signaling pathway activation was evaluated by western blot. We found that among 16 lncRNAs which might interact with YAP1, lncPTC1 expressed higher in PTC tissues than their paired normal thyroid tissues. Its over‐expression related to larger tumor size, lymph node metastasis and advanced TNM stage. RIP and RNA pull‐down data showed that lncPTC1 can interact with YAP1, we also found an exact RNA combining area of YAP1 by RIP. The results of function experiments suggested that lncPTC1 could induce cell proliferation, migration and inhibit cell apoptosis in PTC cell lines. Western blot assay showed lncPTC1 can increase expression of YAP1 protein and lower phosphorylation level of YAP1 and its upstream proteins Lats and Mst. Our study suggests that YAP1 interacting lncPTC1 is involved in tumorigenesis of PTC, lncPTC1 can stabilize structure of YAP1 protein and influence phosphorylation of proteins on upstream of Hippo pathway.

RISK STRATIFICATION OF PEDIATRIC PATIENTS WITH DIFFERENTIATED THYROID CANCER

O.H. Baz, I. Chelghoume, F. Hasbellaoui, S. Mimouni

Medecine, CPMC, Algiers, Algérie, Algeria

Differentiated thyroid cancer (DTC) is one of the most common cancers in young female patients, and the incidence has been increasing for the last decades. The mortality of pediatric patients DTC was lower than in adult patients despite more extensive disease and recurrent/persistent disease.

The objective of this study was to evaluate the usefulness of American Thyroid Association (ATA) risk classification and dynamic risk stratification (DRS) based on the response to initial therapy in pediatric patients with differentiated thyroid cancer (DTC). Our cohort study included 45 pediatric patients with DTC (10 boys, 33 girls) aged between 05–16 years who underwent thyroid surgery. Clinical outcomes during median of 6.15 years of follow up were assessed according to 3 ATA risk groups and 4 DRS groups. In ATA risk classification, 58%, 13%, and 29% of patients were in the low‐, intermediate‐, and high‐risk groups, respectively. There was no significant difference in disease‐free survival (DFS) between the indeterminate and the low‐risk group. In DRS, 46.66%, 33.33%, 6.66%, and 13.33% of patients were classified in the excellent, indeterminate, biochemical incomplete, and structural incomplete response groups, respectively. The risk of recurrent/persistent disease was significantly higher in the indeterminate group and in the structural incomplete group compared with the excellent response group. Many staging systems have been defined for DTC which all share the same aim. DRS based on the response to initial therapy could be useful in addition to initial ATA pediatric risk classification to predict recurrent/persistent disease in pediatric patients with DTC.

Of course, risk stratification is not a goal per se. The eventual goal of risk estimation is to separate those patients who require more intensive monitoring from those who can be put at ease and left to a less frequent and less intensive follow‐up. Long‐term follow‐up studies of pediatric DTC cohorts are necessary to increase our current knowledge of pediatric DTC.

PLEURAL EFFUSIONS IN METASTATIC THYROID CARCINOMA: A CASE SERIES

D.T. Broome¹, C. Zayouna², N. Joshi³, L. Lam⁴, J. Geiger⁵, C. Nasr¹

¹Department of Endocrinology, Diabetes & Metabolism, Cleveland Clinic Foundation, Cleveland, OH; ²Internal Medicine, Cleveland Clinic Foundation, Cleveland, OH; ³Department of Radiation Oncology, Cleveland Clinic Foundation, Cleveland, OH; ⁴Department of Pulmonary Medicine and Critical Care, Cleveland Clinic Foundation, Cleveland, OH; ⁵Department of Hematology and Oncology, Cleveland Clinic Foundation, Cleveland, OH

In general, follicular cell‐derived thyroid cancers have an excellent prognosis; however, the development of metastases and pleural effusion can significantly reduce survival. In this case series, we describe the findings of 23 patients with metastatic follicular cell‐derived thyroid carcinoma that presented to a tertiary referral center from January 1, 1990 to April, 2019 who developed a pleural effusion. Data were collected until August 26, 2019 and descriptive statistics were reported with frequencies and ranges. There were 14 male (60.9%) and 9 female (39.1%) patients, of which 18 were white (78.3%), 4 were black (17.4%), and 1 (4.3%) patient without a reported race. The mean age at presentation was 70.4 (range 28.0–89.0) years, and the mean length of follow‐up was 30.8 +/‐ 27.2 months. The most common type of cancer was papillary thyroid carcinoma (69.6%), followed by anaplastic (21.7%), poorly differentiated (8.7%), and follicular thyroid carcinoma (8.7%). The most common histologic subtypes were follicular (39.1%), classical (30.4%), tall cell (26.1%), solid (13.0%), micropapillary (13.0%), oncocytic (8.7%), and insular variants (4.3%). All patients had biopsy‐proven lung metastases, and the most common site was the peripheral/pleural location (73.9%). Of effusions that were sampled, 81.2% were positive for malignant cells. During the study follow‐up period, 19 (82.8%) patients died from disease and only 4 (17.2%) patients survived. When patients developed a pleural effusion, the mean survival was 20.4 (range 0.2–75.3) months. Our study suggests that patients with metastatic follicular cell‐derived thyroid carcinoma who develop a pleural effusion have a poor prognosis.

THE PHENOMENON OF PTH ELEVATION AFTER TOTAL THYROIDECTOMY AND ITS CLINICAL APPLICATIONS

L. Evans¹, A. Young², A. Barber², R. Wang²

¹Otolaryngology, University of Nevada, Reno School of Medicine, Las Vegas, NV; ²Otolaryngology ‐ Head & Neck Surgery, University of Nevada, Las Vegas School of Medicine, Las Vegas, NV

A transient rise in PTH after total thyroidectomy has been reported in the literature, due to an unclear mechanism of action, with unclear clinical significance. Hypocalcemia and hypoparathyroidism remain common post‐operative complications after thyroidectomy. Retrospective cohort of 340 thyroidectomies 2012–2018 at a single institutionSignificant PTH elevations from pre‐op holding were seen after anesthesia induction (p < 0.01) and before thyroid gland removal (p < 0.01), with tapering levels through 20 minutes post‐excision. 96 patients showed elevated PTH from baseline at 20 minutes post‐excision; none of these patients exhibited hypocalcemia symptoms, need for calcium supplementation, corrected calcium levels less than 7.5 mg/dL, or PTH <15 during post‐operative day (POD) 1. Midnight and 6am calcium levels post‐operatively were higher among those with PTH elevation at 20 minutes than those without PTH elevation (p = 0.027; p = 0.003). POD 1 PTH levels were also significantly higher in those who had shown the 20‐minute elevation from baseline as well (p < 0.001). When evaluating for any elevation at 20 minutes post‐excision compared to pre‐op, no significant difference was observed between total, completion, and hemithyroidectomy (p = 0.16); but when using criteria of 2 or more S.D. above baseline, a significant difference was seen (p < 0.001) with total thyroidectomy having less instances of elevation and hemithyroidectomy more. Elevations of PTH at 20 minutes post‐excision predicts normal or elevated POD 1 PTH, no hypocalcemic symptoms, and the lack of need for calcium supplementation in all patients in our series. The observation that 2 S.D. elevations are significantly more often found in hemithyroidectomies (vs total) may reflect that undamaged parathyroid glands (at least 2 in most hemithyroidectomy cases) respond to anesthetic/surgical stress more robustly compared to that in total in which up to 4 glands may be damaged to some extent. Post‐operative PTH elevation at 20 minutes post‐excision may be useful as a clinical predictor or hypocalcemia and hypoparathyroidism after thyroidectomy.

A RARE CASE OF NUT MIDLINE CARCINOMA IN THE THYROID

Z. Liu^1,2, H. Zhang², P. Su², X. Hao², B. Han^1,2

¹Department of Pathology, Shandong University, School of Basic Medical Sciences, Jinan, China; ²Department of Pathology, Qilu Hospital of Shandong University, Jinan, China

Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare and aggressive subtype of squamous carcinoma that typically arises from midline supradiaphragmatic structures, frequently from the head and neck area. It is uniquely driven by the rearrangement of the NUT gene on chromosome 15q14. There has been no previous report of NMC originated from the thyroid. Here, we firstly describe a case of NMC originating in the left lobe of the thyroid of a 38 year‐old male. The patient underwent a laryngectomy and was diagnosed with moderately differentiated squamous cell carcinoma two months ago. Thyroid nodular lesion was discovered on postoperative reexamination of laryngeal carcinoma. Ultrasound showed a 3.8*2.7cm heterogeneous echo mass in the left lobe of the thyroid gland with smooth edges, irregular shape and partial closure of the anterior capsule. Fine needle aspiration cytology smear revealed uniform annular epithelioid cells with scant amphophilic or eosinophilic cytoplasm, and the diagnosis of Bethesda VI, malignancy was rendered. Left thyroidectomy was performed subsequently. Histologically, this case revealed to be poorly differentiated carcinoma consisting of monotonous, medium‐sized round cells with small foci of abrupt keratinization. Immunohistochemistry revealed a strong immunoreaction with NUT, weak immunoreaction with TTF‐1, EMA, CK19, and negative immunoreaction with PAX‐8, TG, CD30, and ALK. Fluorescence in situ hybridization identified a NUT gene isolated from chromosome 15q14. Negative immunoreaction with NUT and TTF‐1 was confirmed in the laryngeal squamous cell carcinoma, which suggested that they are carcinoma from different origination. To the best of our knowledge, this is by far the first case of NUT carcinoma of thyroid origination.

MULTIMODAL THERAPY OUTCOME OF ANAPLASTIC THYROID CANCER : A SINGLE INSTITUTION 10‐YEAR EXPERIENCE IN CHINA

H. Luo¹, L. Zhang¹, Y. Jiang², T. Yang², L. Wang¹, Y. Liu¹, R. Gong¹, T. Wei¹, Z. Li¹, J. Zhu¹

¹Thyroid & parathyroid surgery, West China hospital, Sichuan University, Chengdu, Sichuan, China; ²Department of Pathology, West China hospital, Sichuan University, Chengdu, China

Anaplastic thyroid carcinoma (ATC) is rare but lethal malignancy, with 1 year surviving rate less than 20%. ATA guideline advocated intensive and multimodal therapy regarding ATC treatment since 2012. However, the paucity of evidence reported from China blurs the treatment panorama of Chinese patients. The aim of the study was to analyze the clinical outcomes of patients with ATC in a regional tertiary medical center in China. Retrospectively reviewed the patient charts from 2009–2019. Demographic data, pathological, clinical treatment and follow‐up information was extracted and summarized. Overall survival (OS) and significance were evaluated by Kaplan‐Meier curve and log‐rank test respectively. The IRB of West China hospital proved the study (No.2019‐592).46 ATC patients were enrolled into final analysis with sex ratio 28:18 (female/male) and mean age 63.4 years. Generally, median and mean of OS was 3.1 and 12.8 months respectively. 1‐year and 3‐year OS was 26.10% and 10.87%, respectively. Regarding adjuvant therapy, both radiotherapy (RT) (20.6 vs 1.4 months, p = 0.0105) and chemotherapy (CT) (25.6 vs 1.4 months; p = 0.0014) improved OS. Of note, 7 out of 46 patients treated with multimodal therapy (surgery plus RT and CT) had an impressive survival‐median OS 25.6 months‐768 days. Multivariate analysis identified sex (female) [HR: 3.36; 95% CI (1.1382, 8.17), p = 0.008], multimodal therapy [HR: 8.714, 95% CI (2.23,34.055), p = 0.002], were prognosis predictor. Anaplastic thyroid carcinoma represents a very dismal prognosis. Outcome of multimodal therapy (combination of surgery, RT and CT) suggested optimal option for ATC patients. And multimodal treatment model was independent predictor of better prognosis.

PROBABILITY OF CURE IN DIFFERENTIATED THYROID CANCER (DTC) PATIENTS ACCORDING TO RISK CATEGORIES AND DISEASE STATUS AFTER INITIAL TREATMENT (PERSISTENT OR RECURRENT DISEASE): A SIMPLE PROGNOSTIC MODEL

G. Sapuppo¹, M. Tavarelli¹, R. Masucci², G. Pellegriti³

¹Endocrinology, Department of Clinical and Experimental Medicine, Garibaldi‐Nesima Medical Center, Catania, Italy; ²Department of Surgical Oncology, Garibaldi‐Nesima Medical Center, Catania, Italy; ³Endocrinology, Garibaldi‐Nesima Medical Center, Catania, Catania, Italy

DTC is generally associated with an excellent prognosis, however, up to 20% of patients have disease events during follow‐up, depending on initial prognostic factors and response to initial treatment. We evaluated the probability of cure in a continuous series of 4,292 patients undergone surgery ±¹³¹I treatment for DTC, followed‐up (median 59.4 months; IQR 32.4–111.4) in our Thyroid Clinic. According to risk categories patients were divided in two groups: group A 2427 (56.5%) with very low (unifocal <1cm N0/Nx) and low (multifocal or 1–4cm N0/Nx) risk tumors and group B 1865 (43.5%) intermediate (>4cm or minimal extra‐thyroid extension or N1) and high risk (massive extra‐thyroid extension). According to the response to initial treatment we classified disease as persistent disease (PD)(events within 12 months) and recurrent disease (RD)(after at least 1 year of disease‐free status following initial treatment). During the follow‐up,639/4292 (14.9%) patients presented disease events:210 (8.7%) in group A and 429 (23%) in group B (p < 0.001). Particularly,58.6% in group A and 87.4% in group B had PD (p < 0.001) and 41.4% in group A and 12.6% in group B RD (Fig. A).

At last visit 488 (11.4%) patients were not cured:132 (5.4%) in group A and 356 (19.1%) in group B (p < 0.001) with a percentage of persistence respectively of 75% and 92.1%.

The probability of cure was higher in group A vs group B (37.1% vs 17.0%, p < 0.001); however considering PD and RD separately regardless of risk category data weren't statistically significant (19.5% vs 12.5% in PD, p = 0.07 and 62.1% vs 48.1% in RD, p = 0.1).

Recurrent vs persistent disease had higher probability of cure regardless of initial risk category (62.1% vs 19.5% in groupA, p < 0.001 and 48.1% vs 12.5% in groupB, p < 0.001). Our results show that both risk categories and disease status after initial DTC treatment are useful in predicting the probability of cure during the long‐term follow‐up: in patients with PD this probability is low independently from risk categories (<20%), while in patients with RD it's higher (∼50‐60%). The combination of risk category and disease status represents a simple and useful prognostic model to identify more aggressive DTCs requiring intensive and tailored follow‐up.

PRIOR CANCERS AMONG PATIENTS NEWLY DIAGNOSED WITH ANAPLASTIC THYROID CARCINOMA: PREVALENCE AND IMPACT ON SURVIVAL

J. Sperling¹, A. Maniakas², M. Goswami², G.J. Cote³, R. Dadu³, N.L. Busaidy³, S. Lai², N. Gross², R. Goepfert², E. Sturgis², M.E. Zafereo², M.E. Cabanillas³, J.R. Wang²

¹UTRGV School of Medicine, McAllen, TX; ²Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, TX; ³Department of Endocrine Neoplasia and Hormonal Disorders, The University of Texas MD Anderson Cancer Center, Houston, TX

Approximately 20% of patients with anaplastic thyroid cancer (ATC) report a history of a prior differentiated thyroid carcinoma (DTC). However, prevalence of prior non‐thyroid cancers has not been systematically reported in the literature. We examined the prevalence and survival of ATC patients with a prior history of non‐thyroid cancer and non‐medullary thyroid carcinoma. We identified 466 patients with ATC treated at MD Anderson Cancer Center between February 2000 and March 2019. History of non‐thyroid cancer and thyroid carcinoma were ascertained through retrospective chart and pathology review. Eight patients without histopathological confirmation or adequate follow‐up data were excluded from analysis. Survival analysis was conducted using Kaplan‐Meier's method. The median age of ATC diagnosis was 66 years (range = 25–96 years). A reported prior history of non‐thyroid cancer was found in 102 patients (23%), including 15 (15%) with two or more prior cancers. These varied to include common cancers, rare cancers and hematological malignancies. The most common prior non‐thyroid cancers were skin, prostate, and breast. Median follow‐up time from prior non‐thyroid cancer diagnosis to ATC diagnosis was 8 years (range = 0.5–53 years). A prior history of thyroid carcinoma was reported in 63 patients (14%), and median follow‐up time from prior thyroid carcinoma diagnosis to ATC diagnosis was 3 years (range = 0.5–35 years). Prior thyroid cancers included papillary (79%), follicular (11%) and Hurthle (5%) carcinomas. On pathology, a concomitant DTC component was noted in 233 patients (51%). 35 (15%) of these patients had a reported prior clinical diagnosis of their DTC. Log‐rank analysis showed no significant difference in survival of ATC patients with a prior history of non‐thyroid cancer (p = 0.352) or thyroid carcinoma (p = 0.429) compared to those without reported prior cancer history. Prior non‐thyroid cancers were prevalent in ATC patients. Rates of prior thyroid carcinoma were similar to previous studies, but a higher percentage of patients were found to have concomitant DTC on pathology. A prior history of non‐thyroid cancer or thyroid carcinoma did not appear to significantly impact survival in ATC patients.

THE DIAGNOSTIC VALUE OF CONTRAST ENHANCED ULTRASOUND IN THYROID NODULES THAT CLASSIFIED TO INTERMEDIATE AND LOW RISK ACCORDING TO 2015 ATA GUIDELINES

X. XI², G. Luying¹, Z. Bo²

¹Ultrasound, Peking Union Medical College Hospital, Beijing, China; ²China‐Japan Friendship Hospita, Beijing, China

The diagnostic value of contrast enhanced ultrasound in thyroid nodules that classified to intermediate and low risk according to 2015 ATA GuidelinesConsecutive patients who underwent thyroidectomy between 2011 and 2019 were included. All patients were received conventional ultrasound and CEUS examinations before surgery. The thyroid nodules were classified according to the stratification of ATA guidelines. The analysis included 147 intermediate and low risk thyroid nodules. Heterogeneous enhancement pattern (OR = 7.339, p<0.001) and the relative delayed arrival time of microbubble (OR = 3.209, p = 0.042) were independent risk factors in the diagnosis of intermediate and low risk thyroid nodules. And the sensitivity, specificity and accuracy of heterogeneous were 58.3%, 87.8% and 83.0%, respectively. Among 58 intermediate risk nodules, there had difference in the relative arrival time of microbubble, peak intensity and enhancement pattern between benign and malignant groups (p < 0.05). The sensitivity, specificity and accuracy of heterogeneous were 61.9%, 83.8% and 75.9%, respectively. Among 89 low risk thyroid nodules, there were no significant difference in the relative arrival of microbubble, peak intensity and enhancement pattern between benign and malignant groups. Ring enhancement pattern combined with 2015 ATA guidelines help to reduce the fine‐needle Aspiration (FNA) rate of low risk thyroid nodules, and 86.8% FNA would be avoided for low risk nodules. Contrast enhanced ultrasound plays an important role in diagnosing benign and malignant thyroid nodules that classified into intermediate and low risk. Ring enhancement could reduce the rate of fine needle biopsy in low risk thyroid nodules.

EVALUATED THE VALUE OF ULTRASONOGRAPHIC MULTIMODALITY DIAGNOSTIC MODEL OF THYROID NODULES

X. XI², G. Luying¹, Z. Bo²

¹Ultrasound, Peking Union Medical College Hospital, Beijing, China; ²Department of Ultrasound, China‐Japan Friendship Hospital, Beijing, China

The purpose of this study was to established an ultrasonographic multimodality diagnostic model and verified its efficacy. Consecutive patients who underwent thyroidectomy between 2011 and 2019 were included. All patients were received conventional ultrasound, contrast enhanced ultrasound (CEUS) and strain elastography (SE) examinations before surgery. Independent ultrasound features associated with malignancy were identified using multivariate regression, and to build a diagnostic model based on the ultrasound risk features and the diagnostic value of this model was verified. The analysis included 604 nodules (502 patients, 44.8 ± 0.5 years, 369 women and 133 men): 213 were benign and 391 were malignant. Multivariate regression revealed the following independent risk factors for thyroid cancer: hypoechoic/very hypoechoic, irregular/lobulate margin, microcalcification, irregular blood flow distribution and heterogenous enhancement. Ultrasonographic multimodality diagnostic model was established: P = , z = ‐3.684 + 1.702 × heterogenous enhancement +1.686 × hypoechoic or very hypoechoic +1.221 × microcalcification +1.107 × irregular or lobulated margin +0.994 × irregular blood flow distribution. When p > 0.60 diagnosed as malignant, this model has high diagnostic value, the sensitivity, specificity and accuracy were 89.7%, 81.8% and 86.9%, respectively. The specificity and accuracy and AUC value were higher than any other single mode (conventional ultrasound, CEUS or SE) (p < 0.05). Ultrasonographic multimodality diagnostic model was verified by using 72 thyroid nodules and the results indicated that this model has high diagnostic value for benign and malignant thyroid nodules. Ultrasonographic multimodality diagnostic model has high diagnostic value for benign and malignant thyroid nodules, and can improve the diagnostic accuracy of thyroid cancer compared with any single mode.

TESTING OF APPROVED DRUGS ON HUMAN DIO1 INTERFERENCE – A SCREENING APPROACH

K. Renko, N. Wiese, J. Goehmann, C. Frädrich, J. Köhrle

Charite‐Universitätsmedizin Berlin, Berlin, Germany

Development and application of novel drugs frequently fails due to their interference with the thyroid gland, thyroid hormone (TH) binding proteins, TH transporters, or TH metabolism. Toxicologists distinguish between ‘direct’ and ‘unspecific’ interference, implying drug induced enzyme induction resulting in ‘secondary’ increased TH elimination from circulation. Direct inactivation of hepatic DIO1 activity by drugs is rarely investigated but commonly used drugs may influence TH status via this mechanism.

Based on our high throughput screening (HTS) platform using the Sandell‐Kolthoff reaction, we tested this hypothesis with a collection of FDA‐approved drugs. Human recombinantly expressed DIO1 activity and hepatic S9 fraction expressing endogenous DIO1 were used as enzyme sources to increase human toxicological relevance. DIO1 activities from both sources were determined by our non‐radioactive method in presence/absence of test drugs. In an initial screening using HEK293‐DIO1, 1953 ‘blinded’ components were tested at a fixed 20μM final concentration in duplicates. For the most potent components (>50% inhibition) IC50 values were verified in dilution series complementary with DIO1 and S9 fraction. At fixed 20 μM concentration, 3,7% of tested drugs showed >25% DIO1 inhibition and 1,3% an inhibition of >50%. Among them were known DIO1 inhibitors (PTU, Genistein) supporting the high predictive quality of the screening.

In comparative verification of 27 most potent compounds, 10 lost inhibition in S9 fraction, indicating their metabolic inactivation, while 17 revealed 1–100μM IC50 values. IC50 determinations with recombinant DIO1 enzyme confirmed all initial screen hits. Results show that among FDA approved drugs several substances may exert specific and potent effects on extrathyroidal TH metabolism. Apart from such influence on circulating TH, their intracellular effects need to be examined. A more detailed survey of their side effects, combined with their analysis using intact cells and a review of their isoenzyme specificity will reveal to what extent other adverse effects (e.g. induction of fatty liver) could mechanistically be related to the influence of commonly used drugs on hepatic deiodinase activities.

DIAGNOSTIC ACCURACY AND UTILITY OF COMPUTER‐AIDED DIAGNOSIS SYSTEM FOR THYROID NODULES ON ULTRASONOGRAPHY

Q. Gao¹, B. Zhang²

¹Department of Ultrasound, Peking Union Medical College Hospital, Beijing, China; ²Department of Ultrasound, China‐Japan Friendship Hospital, Beijing, China

To evaluate diagnostic performances of a computer‐aided diagnosis(CAD)system for thyroid nodules on ultrasonography and the integration of the CAD system with radiologists who have different level of experience in ultrasonography. A total of 158 thyroid nodules (58 benign,100 malignant) of 117 patients between September 2017 and March 2018 were evaluated retrospectively. Two radiologists (an inexperienced radiologist who is a ultrasonography resident and an experienced radiologist who has 10 years of experience in ultrasonography) and the CAD system to analyze ultrasonography images independently, then two radiologists gave a final diagnosis after being informed of the analytic results of the CAD system. We compared diagnostic performances of the CAD system, radiologists and the integration of the CAD system with radiologists. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the CAD system were 90.00%,72.41%,84.91%,80.77%,83.54% respectively. Compared with two radiologists, the CAD system had the highest sensitivity and area under the receiver operating characteristic curves(AUROC), although the difference was not statistically significant(sensitivity:90.00% vs. 86.00%, p = 0.480,85.00%, p = 0.267; AUROC:0.905 vs. 0.862, p = 0.252,0.894, p = 0.736). The specificity of the CAD system was significantly lower than that of the experienced radiologist(72.41% vs. 87.93%, p = 0.016). When the results of the CAD system were integrated, the sensitivity, specificity, accuracy and AUROC of two radiologists had improved, but there were no statistical difference. The CAD system had high value in differentiating malignant thyroid nodules from benign ones and it could become a supplementary method for diagnosing thyroid nodules.

OUTDOOR AIR POLLUTANTS IS A POTENTIAL RISK FACTOR FOR THE INCREASING PREVALENCE OF THYROID NODULE: RESULT FROM A 4.9 MILLION CHINESE POPULATION

S. Wang¹, Y. Song², Q. Wei⁵, C. Jin^3,4, M. Li¹, Y. Ning^3,4, L. Li¹

¹Epidemiology&Bio‐statistics, Peking University Health science center, Beijing, China; ²Department of Endocrinology and Metabolism, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, China; ³PKUHSC Meinian Public Health Research Institute, Beijing, China; ⁴Meinian Institute of Health, Beijing, China; ⁵Department of Social Medicine and Health Education, Beijing, China

Outdoor air pollutants, including PM_2.5, PM₁₀, NO₂, SO₂, CO, and O₃, are known risk factors for cardiovascular diseases and cancer, but their associations with the thyroid nodule which are highly prevalent worldwide, have not been well explored. We aim to investigate relationships between air pollutants and thyroid nodule in China. We utilized a database including participants from a health checkup population who attended routine physical examinations in Meinian HealthCare Screening Center in 157 Chinese cities in 2017. A total of 4.9 million enrollers aged ≥18 year old were included in analysis. Average City‐level annual concentrations of air pollutants from 2015 to 2017 were calculated based on data from China's National Urban Air Quality Real Time Publishing Platform. Logistic regression was used to examine the associations with adjustment for age, sex, education, smoking, body mass index, fasting blood glucose, triglyceride, low density lipoprotein cholesterol, high density lipoprotein cholesterol, urine iodine, gross domestic product, and thyroid stimulating hormone, and O₃ for PM_2.5, PM₁₀, NO₂, SO₂, CO. We used stratified analyses to investigate effect modification by sex, age, and urine iodine groups. Statistically significant associations were detected for any of six air pollutants and thyroid nodule in adjusted models. The odds ratios (OR, 95%CI) were 1.03 (1.03, 1.03), 1.02 (1.02, 1.03) for PM₁₀, 1.01 (1.01, 1.02) for NO₂, 1.09 (1.09, 1.10) for SO₂, and 1.13 (1.12, 1.13) for 10 μg/m³ increase for PM_2.5, PM₁₀, NO₂, SO₂, and O₃. The OR (95%CI) estimate was 1.40 (1.37, 1.42) per mg/ m³ for CO. Nonlinearity tests indicated that all these associations did not deviate significantly from a linear association. Stratified analysis indicated that ORs were relatively lower in participants who were women, elder, as well as having higher urine iodine level. This large cross‐sectional study found liner associations between PM_2.5, PM₁₀, NO₂, SO₂, CO, O₃ and thyroid nodule without minimum effective concentration in China.

CHARACTERISTICS OF CYTOLOGICALLY INDETERMINATE, MOLECULARLY BENIGN NODULES WHICH UNDERWENT SURGERY

C. Long, M. Endo, K. Porter, K. Roll, J.A. Sipos, F. Nabhan

Ohio State University, Columbus, OH

Seventeen percent of benign thyroid nodules will ultimately undergo thyroidectomy. There are no known clinical factors to predict which nodules will eventually require surgery. The aim of this study is to investigate baseline clinical characteristics of cytologically indeterminate, molecularly benign thyroid nodules which subsequently underwent surgery, to assess for predictive factors of eventual removal.

This was a retrospective analysis of all Bethesda III/IV nodules with benign Afirma molecular testing from 2/2011 to 12/2018 at The Ohio State University. Statistical analysis was performed with chi‐square test for categorical values and Mann‐Whitney U test for continuous unpaired values. Two‐sided P values of less than 0.05 were considered statistically significant.

Of all 296 nodules, 30 underwent thyroidectomy and all had benign surgical pathology. The reason for surgery was compressive symptoms in 40% of patients, presence of other suspicious nodules in 37%, and patient preference in 3%. The average time between biopsy and surgery was 299 days. Statistically significant nodule growth was seen in the surgical group compared to unoperated counterparts (+0.35 cm v.s ‐0.01cm, p = 0.01). Patients who underwent surgery were significantly younger at the time of biopsy (50 vs 55 years, p = 0.02). FDG‐PET avidity was higher in surgical group who had PET testing (83% v.s 65%, p‐0.51). There was no significant difference in nodule size, gender, the prevalence of Hurthle cell changes, Bethesda III and Bethesda IV results.

Younger age at initial biopsy and nodule growth was associated with a higher likelihood of surgical intervention in cytologically indeterminate, molecularly benign thyroid nodules.

IMPACT OF BODY MASS INDEX ON REMOTE‐ACCESS THYROIDECTOMY: EXPERIENCE WITH ROBOTIC TRANSAXILLARY AND TRANSORAL ENDOSCOPIC VESTIBULAR APPROACHES

R. Liu¹, M.A. Razavi², C.R. Razavi¹, M. Shalaby², M. Shaear¹, R.P. Tufano¹, E. Kandil², J. Russell¹

¹Otolaryngology‐Head and Neck Surgery, Johns Hopkins University School of Medicine, Baltimore, MD; ²Surgery, Tulane University School of Medicine, New Orleans, LA

Remote‐access approaches (RAA) in thyroid surgery have garnered international attention as an alternative to open surgery for patients who wish to avoid an anterior cervical scar. Although international data has demonstrated the safety of RAAs, questions regarding generalizability, given the body habitus of the North American population, remain. Here we aim to characterize the safety profile of two remote‐access approaches ‐ the transoral endoscopic vestibular thyroidectomy approach (TOETVA) and the robotic transaxillary approach (RTA) ‐ in a North American population, particularly with regards to the impact of body mass index (BMI) on outcomes, and in the context of the conventional transcervical approach (TCA) for this patient population. Cases of TOETVA, RTA, and TCA thyroidectomy (meeting criteria for RAA) were reviewed over a 2‐year period across two North American academic medical institutions. Primary outcomes were postoperative complications, which were compared between approach cohorts (TOETVA, RTA, and TCA) and within approach cohorts across NIH‐defined BMI classes (underweight/normal <25 kg/m², overweight 25–29.9 kg/m², and obese ≥30 kg/m²).118 TOETVA, 133 RTA, and 259 TCA cases were reviewed. Operative times were significantly longer for RAA cohorts than for the TCA cohort for both lobectomies (TOETVA 133 minutes; RTA 130 minutes; TCA 87 minutes; p = 0.0001) and total thyroidectomies (TOETVA 180 minutes; RTA 166 minutes; TCA 118 minutes; p = 0.0001). There were no differences in complication rates across the approach cohorts (TOETVA 16.1%; RTA 8.3%; TCA 13.5%; p = 0.14), including recurrent laryngeal nerve injury and transient hypoparathyroidism. There were also no differences in complication rates with respect to BMI groups within the TOETVA (13.6%; 16.7%; 18.2%; p = 0.86) and TCA (15.8%; 10.3%; 14.2%; p = 0.63) cohorts. The RTA cohort did not have any complication within the obese group (8.3%; 15.4%; 0%; p = 0.04). RAAs, specifically TOETVA and RTA, can be performed safely in a select North American patient population. As higher BMI did not lead to increased risk for complications, higher BMI should not preclude candidacy for remote‐access thyroidectomy.

PERFORMANCE OF THYROSEQ GENOMIC CLASSIFIER IN CYTOLOGICALLY INDETERMINATE THYROID NODULES IN SOUTHEAST ASIAN POPULATION WITH HIGH PRE‐TEST CANCER PROBABILITY

S.P. Yang¹, M. Nga², S. Tan³, J. Lum², R. Parameswaran⁴, K. Loh⁵, X. Huang⁶, M. Lim⁷, T. Ho⁸, S. R.M⁹, W. Tan⁴, K. Ngiam⁴, H. Li⁷, A.D. Rao⁹, M.M. Bundele¹⁰, V. Desai¹¹, K. Lim⁷, A. Tan¹, K.S. Sek¹, W. Cheah¹², N. Rao¹⁰, D.C. Chin⁶, D.Y. Chua⁷, A.I. Wald¹³, L.A. Tolino¹³, T. Tun¹, E. Tai¹, Y.E. Nikiforov¹³

¹Endocrine Medicine, National University Hospital, Singapore, Singapore; ²Department of Pathology, National University Hospital, Singapore, Singapore; ³Department of Pathology, Changi General Hospital, Singapore, Singapore; ⁴Department of General Surgery, University Surgical Cluster, National University Hospital, Singapore, Singapore; ⁵Department of Otolaryngology – Head and Neck Surgery, National University Hospital, Singapore, Singapore; ⁶Department of Otolaryngology, Head and Neck Surgery, Changi General Hospital, Singapore, Singapore; ⁷Department of Otorhinolaryngology, Tan Tock Seng Hospital, Singapore, Singapore; ⁸Department of General Surgery, Tan Tock Seng Hospital, Singapore, Singapore; ⁹Endocrine Surgical Unit, Department of Surgery, Khoo Teck Puat Hospital, Singapore, Singapore; ¹⁰Department of Pathology, Tan Tock Seng Hospital, Singapore, Singapore; ¹¹Department of Pathology, Khoo Teck Puat Hospital, Singapore, Singapore; ¹²Department of General Surgery, Ng Teng Fong General Hospital, Singapore, Singapore; ¹³Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA

Introduction: The difficulty in making a definitive cytologic diagnosis in indeterminate thyroid fine needle aspirates (FNAs) leads to suboptimal patient management with significant rate of avoidable diagnostic surgeries and two‐stage thyroidectomies. The performance of FNA molecular testing in Asian populations is not well studied. The aim of this study was to evaluate the performance and potential clinical utility of ThyroSeq Genomic Classifier (GC) in cytologically indeterminate thyroid nodules in Singapore.

Methods: A multicenter, prospective, blinded study was conducted in 4 tertiary hospitals, recruiting consecutive patients with thyroid FNAs of nodules falling into the Bethesda categories III (n = 61, 63.0%), IV (n = 17, 17.5%), and V (n = 19, 19.5%), which underwent surgical excision. FNA needle washing were used to perform ThyroSeq GC analysis. The results were correlated with corresponding histology.

Results: Out of 98 thyroid FNAs, 97 (99%) had sufficient material for testing. Cancer prevalence (including 2 NIFTP cases) on histology was 33%, 41%, and 84% in Bethesda III, IV, and V nodules, respectively. ThyroSeq yielded negative test result in 41% of cases and positive result in 59%. The sensitivity, specificity, NPV and PPV in the entire cohort were 90%, 65%, 90%, and 67%, respectively. Findings included 15 BRAF V600E, 5 TERT mutations, and 2 NTRK fusions. The test correctly identified medullary thyroid carcinoma and parathyroid adenoma. The false negative rate was 4% (n = 4). The false positive rate was 20% (n = 19) involving mainly RAS‐like nodules (15 RAS, 2 with THADA‐IGF2BP3 fusion, 1 EIF1AX, and 1 with copy number changes). The true positive rate of 26 RAS‐mutants in whole cohort was 42%, and that of the RAS‐mutants with nuclear atypia was 45%.

Conclusions: The high NPV of ThyroSeq test could have potentially avoided unnecessary diagnostic thyroidectomy in 41% of patients with indeterminate nodules even in populations with high pre‐test cancer probability. Of nodules tested positive, the risk of malignancy ranged 50 to 99%. Nodules with molecular profile predicting high‐risk of malignancy may be suitable for total thyroidectomy to avoid two‐stage thyroidectomy.

PARATHYROID ALLOTRANSPLANTATION AS A DEFINITIVE CURE FOR HYPOPARATHYROIDISM: AN HISTORIC REVIEW.

B.R. Rosvall, D.W. Cote

University of Alberta, Edmonton, Alberta, Canada

Hypoparathyroidism is the most common complication following total thyroidectomy, occurring in 1.5% to 2.5% of patients. Permanent hypoparathyroidism requires lifelong medical maintenance treatment to prevent life‐threatening conditions such as seizure, cardiac arrhythmia, and congestive heart failure. Currently, there is no widely accepted cure for the condition. A review of the literature was performed to assess the historic use and modern advancements of parathyroid allotransplantation as a definitive treatment for hypoparathyroidism. In 1911, Brown reported the first parathyroid allotransplant. It was not until 1973 that histological proof of parathyroid allotransplant survival was first reported in patients on immunosuppression for renal transplant.

In 2016, the first successful parathyroid allotransplantations were performed from healthy living donors. Neither donors nor recipients experienced complications, suggesting retrieval of parathyroid glands from healthy donors is feasible and safe.

While immunosuppression strictly for parathyroid allotransplantation has been reported, the side effects of immunosuppressants are traditionally viewed as more harmful than medical management. Current research has focused on reducing need for immunosuppression through techniques such as microencapsulation, cryopreservation, and immunologic monitoring. For many tissues, donor and recipient compatibility is assessed pre‐ and post‐transplant, with immunologic tests dependent on tissue type; however, no standard criteria for immunologic monitoring have been developed for parathyroid allotransplantation. Current literature shows promise in pre‐evaluation of compatible parathyroid donor and recipient with panel reactive antibody screening, flow cytometric cross match tests for T and B cells, and microlymphocytotoxicity tests. In patients requiring lifelong immunosuppression, parathyroid allotransplantation offers a potentially curative treatment option associated with limited risk and morbidity. Further research is needed to optimize donor and recipient immunologic compatibility so as to minimize need for immunosuppression.

INFLUENCE OF RAPID URBANIZATION ON THYROID AUTOIMMUNE DISEASE IN CHINA

B. HAN

Shanghai Ninth People's Hospital, Shanghai, China

The prevalence of autoimmune thyroid diseases (AITDs), especially Hashimoto's thyroiditis (HT), has increased dramatically in China. Moreover, China is experiencing the largest scale of urbanization in the world. However, few studies have focused on the relationship between rapid urbanization and HT. A total of 2946 subjects in Zhejiang Shangyu (SY) (n = 1546) and Jiangsu Nanjing (NJ) (n = 1400) were enrolled in this study. Serum TPOAb, TGAb and thyrotropin (TSH) were measured, and ultrasonography of the thyroid was performed in all subjects. DNA was extracted from all subjects, and four SNPs were selected for genotyping. Generalized multifactor dimensionality reduction (GMDR) was used to screen the best interaction between genetic factors and environment factors. TPOAb and TGAb concentrations were higher in NJ than in SY (117.82 ± 298.70 vs. 72.32 ± 247.47 and 57.10 ± 124.89 vs. 25.37 ± 60.34). Residents in NJ had higher TSH levels than those in SY (3.13 ± 5.55 vs. 2.38 ± 1.46). People in NJ also had higher TPOAb and TGAb positivity rates than those in SY (7.8% vs. 12.7% and 8.7% vs. 16.3%). Logistic regression analysis indicated that rapid urbanization was an independent risk factor for TPOAb (OR = 1.473) and TGAb (OR = 1.689), even after adjusting for age, sex, smoking status, BMI and diabetes. Genotype TT in rs11675434 was associated with an increased risk of TPOAb positivity both in SY (OR = 2.955) and in NJ (OR = 1.819). GMDR analysis showed a two‐locus model (SNP2 × urbanization) and a three‐locus model (SNP2 × SNP3 × urbanization), which had testing accuracies of 56.88% and 57.25%, respectively (P values were 0.001 and 0.001). Rapid urbanization influences the incidence of TPOAb and TGAb positivity. Furthermore, rapid urbanization could interact with TPOAb susceptibility genes and promote the increase in TPOAb positivity. We should pay more attention to thyroid autoimmune disease in areas of China experiencing rapid urbanization.

LOW REVERSE T3 : A RELIABLE, SENSITIVE AND SPECIFIC IN DIAGNOSIS OF CENTRAL HYPOTHYROIDISM

U.M. Kabadi^1,2

¹Endocrinology, Broadlawns Medical Center, Des Moines, IA; ²Endocrinology, University of Iowa, Iowa City, IA

Low free T4 and normal/low TSH concentrations are frequently noted in clinical practice and denote presence of either ‘Euthyroid Sick Syndrome’ or Central Hypothyroidism. Moreover, distinguishing one from another is difficult even with determination of free T3 being low/ normal in both disorders. Reassessment of repeated determination of these tests may help differentiate between these disorders. However, precious time is lost.

Objective: Evaluation of serum reverse T3 concentration to differentiate between Central Hypothyroidism and ‘Euthyroid Sick Syndrome’. Serum Free T3 and Reverse T3 concentrations were determined as ‘add on’ tests using previously drawn blood samples of 45 consecutive adult subjects with age, 28–69 years, 24 men and 21 women showing low serum free T4, 0.82 ± 0.02 (normal range, 0.89‐ 1.70 mcg/ dl) and low/ normal TSH, 1.39 ± 0.48 (normal range, 0.45–4.67 uU/ml) concentrations during 12 months, 7/1/2018 ‐ 6/30/2019. Serum free T4, free T3, TSH and reverse T3 levels were also determined in 35 healthy volunteers, 15 men and 20 women. Statistical analyses for comparisons between groups were conducted using Student's ‘t’ test and ANOVA. All data are reported as Mean ± SEM. Serum reverse T3 determination established two distinct groups one with subnormal concentrations, 8.78 ± 0.55 (normal range, 11‐ 26 ng/dl) and another with high normal or supernormal levels, 11.‐35 ng/dl. Serum free T3 concentrations varied between subnormal and normal concentrations, 1.8–2.9 (normal range, 2.3–4.2ng/ml) in both groups established by reverse T3. Moreover, on reassessment after 3–6 weeks, free T4, free T3, TSH and reverse T3 levels normalized in the group with normal or elevated reverse T3 indicative of ‘Euthyroid Sick Syndrome’ whereas free T4 (0.85 ± 0.02 and reverse T3 (8.65 ± 0.49) remained subnormal in the other group suggesting presence of Central Hypothyroidism. Serum reverse T3 concentration is a reliable laboratory test differentiating between Central Hypothyroidism and ‘Euthyroid Sick Syndrome in subjects with low free T4 and Low/ normal TSH levels.

MOLECULAR TESTING OF BRAF MUTATION IN PAPILLARY THYROID CANCER WITH FINE‐NEEDLE ASPIRATION BIOPSY FROM THYROID NODULES BY DROPLET DIGITAL PCR

G. Fu^1,2, R.S. Chazen¹, C. MacMillan^1,3, I. Witterick^1,2

¹Alex and Simona Shnaider Research Laboratory in Molecular Oncology, Department of Medicine, Endocrine Division, Mount Sinai Hospital, Toronto, Ontario, Canada; ²Department of Otolaryngology – Head & Neck Surgery, University of Toronto, Toronto, Ontario, Canada; ³Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada

Thyroid cancer is one of the most rapidly increasing carcinomas and up to 90% of cases are papillary thyroid cancer (PTC). The ultrasound‐guided fine needle aspiration (FNA) cytology is the routine approach to screen thyroid nodules and diagnose PTC. However, approximately 20% of FNAs of nodules are indeterminate. BRAF V600E mutation is frequently associated with PTC occurrence, progression and its poor clinicopathological characteristics and thereby becomes a promising molecular marker to detect PTC. There is a clinical need to develop a novel method to detect BRAF mutation using a small amount of the tumor cells obtained by FNA. This study aimed to develop molecular testing of BRAF mutation in surgical tissues or FNA biopsies from thyroid nodules using droplet digital PCR (ddPCR). Genomic DNA was prepared from surgical tissues or FNA biopsies obtained from 114 patients with thyroid nodules using the QIAGEN DNA Mini Kit (QIAGEN, Germany), including 84 (74%) PTC tumors, 1 (1%) follicular thyroid carcinoma (FTC), 1 (1%) medullar thyroid carcinoma (MTC), 6 (5%) NIFTP and 23 (19%) benign nodules, according to the clinical practice guidelines of Thyroid Carcinoma. Molecular testing with ddPCR was performed to detect BRAF mutation. Genomic DNA from FTC 133 and BCPAP cells was used to optimize ddPCR conditions to detect the wildtype BRAF or mutant BRAF V600E as the negative and positive controls, individually. The linearity of ddPCR detecting BRAF mutation was calculated (y = 1.102 × ‐1.6842, R² = 0.9968) with a high reproducibility (CV = 4.2%) and a sensitivity at 0.92 (0.35–1.35) copies/μl in sample with 1 ng of BRAF mutation. Through ddPCR, BRAF mutation was revealed in 28 (33%) of 84 PTC patients, including 21 (37%) of 56 classic PTC, 1 (5%) of 22 follicular variant PTC and 6 (100%) of 6 tall cell papillary carcinomas, with 100% specificity, 33.3% sensitivity and 100% PPV, however, BRAF mutation was not detected in all benign nodules and NIFTPs. Our results demonstrated that ddPCR can be used in detecting BRAF mutation to identify PTC from FNA samples of thyroid nodules. Ancillary molecular testing with additional markers, such as RAS and TERT promoter mutations, might assist in differentiating NIFTP from PTC.

MULTIPLE LESIONS TRACKING FOR PROGRESSION OF THYROID CANCER LUNG METASTASES

P. Cheng², C. Wright³, F. Nabhan⁴, C. Menq², S.M. Jhiang¹

¹Physiology and Cell Biology, The Ohio State University, Columbus, OH; ²Dept. of Mechanical and Aerospace Engineering, The Ohio State University, Columbus, OH; ³Radiology, The Ohio State University, Columbus, OH; ⁴Endocrinology, The Ohio State University, Columbus, OH

Thyroid cancer patients with lung metastases have variable prognosis, as lung lesions may be indolent or become aggressive with rapid growth rate. Clinical decision is currently guided by monitoring target lesions with unidimensional or bidimensional measurements on cross‐sectional radiologic imaging. We hypothesize that total tumor burden defined by voxel‐based volumetric measurement of detectable lesions along with tumor volume doubling time (TVDT) enabled by lesion tracking can better reflect disease progression status. CT‐Viewer‐lung, an in‐house software, is developed to conduct lesion identification and lesion volume segmentation within the lung region, as well as lesion association between successive CT‐chest images to allow tracking of volume changes in individual lesions as well as total lung lesion burden. De‐identified CT‐chest images (N = 20) were acquired and analyzed from three randomly selected thyroid cancer patients with lung metastasis who have consented to participate in The Ohio State University Endocrine Neoplasia Repository. CT‐Viewer‐lung automatically provided volume calculation for each lesion without subjective manual measurements. It allowed serial monitoring of volumetric progression or regression of individual lung lesions of >20 mm³ regardless of change in their geometry shape and provided longitudinal dynamics in lesion volume and TVDT for individual lesions and total burden of lung lesions. It has detected disease progression earlier than what is reported by radiologists in the standard CT scan reports and before lesions reaching measurable size per RECIST 1.1 definition. There was an observed concordance between changes in total burden of lung lesions and serum thyroglobulin (Tg) levels further attesting to the accuracy of CT‐Viewer‐lung analysis. CT‐Viewer‐lung may assist in clinical decision on monitoring frequency of CT‐chest image for patients under active surveillance, selecting patients for medical intervention, and continuing or altering the therapeutic regimen by evaluating treatment responsiveness.

T CELL RECEPTOR SEQUENCING IDENTIFIES DISTINCT REPERTOIRES OF THYROID MALIGNANT TUMORS AND BENIGN NODULES

X. Lan², Q. Zhang³, X. Ge^4,5, C. Zhang⁶, C. Wang¹, C. Zhang¹, Z. Tai¹, W. Wei¹, J. Cao², Z. Tan², C. Zheng⁷, C. Chen², J. Wang², J. Xu⁷, X. Zhu², M. Ge⁷

¹Geneplus‐ShenZhen, ShenZhen, GuangDong, China; ²Institute of Cancer and Basic Medicine, Chinese Academy of Sciences, Cancer Hospital of the University of Chinese Academy of Sciences, Zhejiang Cancer Hospital, HangZhou, China; ³Zhejiang Chinese Medical University, HangZhou, China; ⁴Institute of Cancer and Basic Medicine, Chinese Academy of Sciences, Hangzhou, China; ⁵Heartland Christian School, Columbiana, OH; ⁶Geneplus‐Beijing, Beijing, China; ⁷Zhejiang Provincial People's Hospital, HangZhou, China

Tumor infiltrating T cell plays an important role in preventing tumorigenesis, suppressing tumor progression and influencing treatment and clinical outcome. Although T cell receptor (TCR) repertoires have been reported in many solid tumors, the comprehensive TCR repertoires of thyroid tumor are still unclear. In this study, we characterized the T cell receptor β‐chains repertoires of 89 thyroid tumor patients using high‐throughput sequencing, which includes 21 benign nodules and 68 malignant tumors. The overall results exhibited that the blood samples have the highest TCRβ diversity (Clonetypes, median = 16120; Shannon index, median = 8.44), and TCRβ diversity of tumor tissues is higher than normal tissues (Shannon index, p < 0.001). Additionally, the TCRβ diversity of malignant tumors is significantly higher than benign nodules in both blood and tumor samples (Shannon index, blood, p < 0.01; tumor, p < 0.001). We further demonstrated that as the malignancy increase, the TCRβ diversity decreased in malignant tumor. The malignant tumors with distal metastasis or invasiveness showed less TCRβ diversity than non‐metastasis (Shannon index, p < 0.01) or non‐invasive (Shannon index, p < 0.01) malignant tumors. Analysis of Morisita‐Horn index indicated that the significant TCRβ repertoires similarity between tumor and blood in distal metastasis thyroid tumors (Comprison with Non‐metastasis, p < 0.05). Finally, based on the distinct TCRβ repertoire differences indicated by multi‐dimensional scaling (MDS) analysis, a support vector machine was used to construct machine learning model to distinguish malignant tumors from benign nodules. The VJ gene combinations and CDR3 amino acid motifs were used to training and fitting the learning model. The validation of both blood and tumor samples showed reasonable performance with the AUC value beyond 0.79. The differential T cell repertoires in malignant tumors and benign nodules suggest a distinct T cell immune microenvironment in different patients. Inspiringly, TCRβ machine learning model with blood sample provide a possibility for non‐invasive diagnosis of malignant thyroid tumor.

AN ACQUIRED NOVEL RAC1^P34R MUTATION DRIVES PROLIFERATION AND ACTIN REORGANIZATION IN PAPILLARY THYROID CANCER CELLS BUT NOT RESISTANCE TO DABRAFENIB

E. McBeath¹, N.L. Busaidy¹, T. Hai¹, B. Danysh¹, M.E. Cabanillas¹, M. Shah², M. Ringel², M. Hoffman¹

¹Endocrine Neoplasia & Hormonal Disorders, MD Anderson Cancer Center, Houston, TX; ²Internal Medicine, Ohio State University, Columbus, OH

BRAF‐activating mutations have a poor prognosis in papillary thyroid cancer (PTC). Resistance develops rapidly to dabrafenib, a targeted inhibitor used for treating advanced PTC, but its mechanisms are unknown. We have identified a novel RAC1^P34R mutation in a BRAF‐mutated PTC patient treated with dabrafenib. As RAC1 is an important regulator of the cell cycle, cell‐cell adhesion, and motility, the objective of this study is to investigate the link between this novel mutation and resistance to dabrafenib treatment. A cell line, FNA001, was derived from a patient sample at progression under dabrafenib. Whole exome sequencing confirmed concomitant BRAF^K601N and RAC1^P34R mutations. FNA001 cell proliferation in the presence of dabrafenib was measured using an IN‐Cell analyzer. RAC1 activation was tested through a GTP‐binding affinity assay. To evaluate the function of mutated RAC1 independently from the BRAF^K601N mutation, we stably expressed wild‐type RAC1 or RAC1^P34R in cell lines that are either BRAF wild type or harbor the BRAF^V600E mutation most commonly observed in PTC. IC50 values for dabrafenib were compared and morphology/actin organization evaluated. RAC1 was highly expressed and activated in FNA001 cells compared to other BRAF‐mutated PTC cells. These cells exhibited a significant ∼7‐fold increase in resistance to dabrafenib, compared to other BRAF‐mutated PTC cells. Stable expression of wild‐type RAC1 or RAC1^P34R in BRAF^V600E cells indicated that excess RAC1 with or without mutation can drive cell proliferation. No increase in dabrafenib resistance was observed in BRAF^V600E; RAC1^P34R‐mutated cells in comparison to BRAF^V600E‐only mutated cells. However, RAC1^P34R induced a significant change in cell morphology and reorganization of F‐actin almost exclusively at the cell cortex. The acquired RAC1^P34R mutation conferred a morphology that might be associated with changes in cell adhesion properties and motility. However, RAC1^P34R was not linked to dabrafenib resistance, which is very likely attributable to the BRAF^K601N mutation alone. Our data underscore the importance of using combination therapies with MEK inhibitors downstream of BRAF mutations to improve the efficacy of treatment in advanced PTC.

THYROID HORMONE MEDIATED TUMOR SUPPRESSION IN ANAPLASTIC THYROID CANCER

E.L. Bolf¹, N.E. Gillis¹, C. Davidson¹, J. Tomczak¹, S. Frietze², F. Carr¹

¹Pharmacology, University of Vermont, Burlington, VT; ²Biomedical and Health Sciences, University of Vermont, Burlington, VT

Thyroid cancer incidence has been steadily increasing over the last several decades, both domestically and internationally, and is now the most commonly diagnosed endocrine cancer. Although differentiated thyroid cancers are highly responsive to existing therapies, anaplastic thyroid cancer (ATC), a dedifferentiated carcinoma, is highly aggressive and unresponsive to current therapies. Median survival after diagnosis is less than 6 months and new treatment options for ATC are needed. Thyroid hormone receptor beta (TRβ) is a nuclear receptor that has been characterized as a potent tumor suppressor in a variety of solid tumors, including in follicular thyroid cancer, however the mechanism that it regulates is not well‐delineated. We seek to better characterize TRβ actions in ATC to explore possible endocrine‐based targets for novel ATC therapies. Expression of TRβ in ATC is low, so we utilized a cell line representative of ATC biology, SW1736, and restored TRβ expression by stable lentiviral transduction. Cell growth was measured by live cell imaging. Cells were grown in serum‐free conditions for tumorsphere formation and anchorage‐independent growth was assessed by a soft agar colony formation assay. RNA‐sequencing was performed to assess the impact of TRβ in ATC, both in the presence and absence of its ligand. Ligand‐activated TRβ reduced the aggressive phenotype as reflected by cell growth, tumorsphere formation, and anchorage independent growth. T₃ activated TRβ induced transcriptomic changes by RNA‐sequencing reflecting a more differentiated phenotype and reduction of oncogenic signaling. Of note, we found evidence of STAT1 activation. STAT1 is a driver of apoptosis and we show that TRβ and T₃ increase pro‐apoptotic signaling in ATC cells. Our results reveal a novel TRβ regulated pathway and a potential ATC vulnerability. Further investigation into TRβ and STAT1 mediated apoptosis could improve the efficacy of existing treatment regimes.

PRDM16, A POTENTIAL BIOMARKER OF PAPILLARY THYROID CANCER, SUPPRESSES EPITHELIAL TO MESENCHYMAL TRANSITION OF PAPILLARY THYROID CANCER CELL LINE

W. Liu, Q. Guan, T. Liao, D. Wen, B. Ma, Q. Ji

Fudan University of Shanghai Cancer Center, Shanghai, China

Papillary thyroid cancer (PTC) comprises the vast majority of thyroid cancer. Although the prognosis of papillary carcinoma is well, there are still some PTC patients with local recurrence and distant metastasis, and the mortality rate is increased. This study mainly explored the expression of PRDM16 gene in human papillary thyroid cancer from the histological level, and further studied the molecular mechanism of PRDM16 gene involved in the development of PTC at the cytological level. Patients (n = 110) who underwent surgery between 2012 and 2017 and was pathologically confirmed as PTC at Fudan University Shanghai Cancer Center (FUSCC) were enrolled. We extracted total RNA of tumor tissues and the paired adjacent thyroid tissues of 110 patients and detected the expression level of PRDM16 gene by qRT‐PCR. We used qRT‐PCR and Western blot to detect the expression level of PRDM16 gene in three PTC cell lines TPC‐1, K1 and BCPAP and one human‐derived normal thyroid follicular epithelial cell line Nthy‐ori 3–1. Based on the overexpression of PRDM16 in three PTC cells, cell proliferation and migration were detected by CCK‐8, Transwell and cell scratch assays, respectively. The related molecular markers of PRDM16 affecting epithelial to mesenchymal transition were screened and verified by qRT‐PCR and Western blot. The expression level of PRDM16 gene in human papillary thyroid carcinoma was significantly lower than that in adjacent tissues (P < 0.001). PRDM16 expression in TPC‐1, BCPAP and K1 PTC cell lines were significantly lower than that of Nthy‐ori‐3 cell line on both the mRNA and protein level. Overexpression of PRDM16 can inhibit the proliferation and migration of PTC cell lines. Overexpression ofPRDM16 can inhibit the epithelial‐mesenchymal transition process of PTC cell lines. The epithelial marker E‐cadherin was upregulated. The mesenchymal molecular markers N‐cadherin, MMP3 and Vimentin were downregulated significantly. This study demonstrates PRDM16 gene is under expressed in PTC. PRDM16 gene can inhibit the migration and proliferation of papillary thyroid cancer cell lines. PRDM16 can inhibit the epithelial‐mesenchymal transition process of PTC cell lines.

TET1 ACCELERATES THYROID CANCER PROGRESSION BY STABILIZING HIF1α VIA CK2/AKT/GSK‐3β SIGNALING AXIS

Q. Yang^1,2, F. Sui¹, X. Li¹, H. Dang¹, B. Ruan^1,2, B. Shi^1,2, P. Hou²

¹Endocrinology, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shannxi, China; ²Key Laboratory for Tumor Precision Medicine of Shannxi Province, Xi'an, China

Tet methylcytosine dioxygenase 1 (TET1) is characterized as DNA demethylase and chromatin structure regulator. Its expression and role in thyroid cancer are still unknown. Hypoxia is a feature of rapid growth in thyroid cancer and associated with poor prognosis. Here, we tried to reveal the role of TET1 in hypoxic thyroid cancer and the underlying mechenism. PTC and normal thyroid tissues were subjected to TET1 expression and 5‐hmC detection. PTC data from TCGA database was referenced. After ectopic expression or knockout of TET1, cancer cell growth and migration were compared with control cells under normoxia and hypoxia. By mating the transgenic mouse, we established BRAF^V600E‐driven PTC harboring different status of Tet1. The body weight curve, thyroid mass, and pathological analysis were performed. The cell lysates and mouse tumor sections were subjected to further studies. PTCs showed slightly lower TET1 levels and decreased 5‐hmC against normal controls. However, PTC patients with higher tumorous TET1 possess shorter survival time. TET1 overexpression inhibited cellular viabilities, and it does the opposite when TET1 was knocked down. However, Tet1 ^‐/‐ PTC mouse were much more healthier than Tet1 ^+/+ mouse, showing gained survival rate, body weights, and thyroid function, and repressed hypoxia inducible factor 1α (HIF1α) expression. Under hypoxia, TET1 facilitated cancer cells to keep rapid growth, which was abolished by HIF1α silence. HIF1α promoted the transcription of casein kinase 2B (CK2β), which under hypoxia, was enhanced by TET1. Increased CK2β then induced PTEN phosphorylation and disability. Therefore, AKT activation and GSK3β repression hindered phosphorylation and ubiquitination‐dependent HIF1α degradation. There is a slight decrease of TET1 in PTC than normal thyroid tissues. However, PTC patients and mouse with high TET1 expression showed worse survival. TET1 in normoxia, prevented thyroid cancer progression, however under hypoxia, it promoted cell growth through stabilizing HIF1α. HIF1α‐driven CK2β transcription was reinforced by TET1. Hence, TET1 accommodated CK2‐PTEN‐AKT‐GSK3β‐HIF1α to form a positive feedback loop under hypoxia, indicating critical role of TET1 in hypoxic cancers.

EFFECTS OF DIFFERENT OPERATIONS ON THE QUALITY OF LIFE OF DIFFERENTIATED THYROID CANCER SURVIVORS WITH LOW‐MEDIAN RISK OF RECURRENCE

W. Chen

The First Affiliated Hospital, Sun Yat‐sen University, Guangzhou, Guangdong, China

Due to the good prognosis of differentiated thyroid cancer (DTC), better quality of life had been taken into account. Total thyroidectomy and lobectomy were dominant surgical types for DTC with low‐median risk of recurrence. This study aimed to compare the health‐related quality of life (HRQoL) of these patients with different operations. This was a prospective observational cohort study, enrolling patients diagnosed with DTC with low‐median risk of recurrence in the First Affiliated Hospital, Sun Yat‐sen University from Oct, 2018 to Mar, 2019. Patients were grouped into Total‐thyroidectomy Group and Lobectomy Group according to the operation they underwent. They were followed up before and 1, 3, 6, 12 months after operation for three questionnaires on HRQoL (EORTC QLQ‐C30), emotional disorders (Hospital Anxiety and Depression Scale, HADS) and disease‐specific symptoms (THYCA‐QoL). This study is ongoing, and data up to Mar, 2019 was analyzed preoperatively and 1, 3 months postoperatively. Of 369 eligible patients, 193 participants underwent lobectomy, while 176 underwent total thyroidectomy, with a higher proportion of larger tumor size, lymph node dissection, RAI and married status. After adjustment for confounders, patients with lobectomy complained less social dysfunction, scar, fatigue, depression, neuromuscular, voice and sympathetic symptoms one month after surgeries. Three months after surgeries, above HRQoL differences between two surgical types decreased gradually, but it remained statistically significant different in sympathetic and fatigue symptoms. Furthermore, at one month follow‐up patients with lobectomy in 1–4cm subgroup had significantly lower scores on fatigue, voice and social dysfunction, but higher scores on insomnia and diarrhea. However, these differences disappeared at three month follow‐up. For DTC patients with low‐median risk of recurrence, those who underwent total thyroidectomy experienced worse HRQoL than those who underwent lobectomy in a short term. Long‐term HRQoL will be observed by additional follow‐up.

RESPONSE TO BRAF WITH/WITHOUT MEK INHIBITORS IN BRAFV600E PLUS TERT PROMOTER MUTATED DIFFERENTIATED THYROID CANCERS (DTC).

S. Fazeli¹, M.K. Gule‐Monroe², M.E. Cabanillas¹, N.L. Busaidy¹, S. Waguespack¹, M.I. Hu¹, C. Jimenez¹, S.I. Sherman¹, M.A. Habra¹, G.J. Cote¹, M. Hoffman¹, R. Bassett³, R. Dadu¹

¹Endocrine Neoplasia and Hormonal Disorders, MD Anderson Cancer Center, Houston, TX; ²Diagnostic Radiology, MD Anderson Cancer Center, Houston, TX; ³Biostatistics, MD Anderson Cancer Center, Houston, TX

Coexisting BRAFV600E and TERT promoter (TERTp) mutations are linked to poor outcomes and worse response to initial treatment in patients (pts) with DTC. The prevalence of the duet in pts who start on targeted therapy (tx) and its impact on response to BRAF and MEK inhibitors (BRAFi/MEKi) have not been studied. This is a retrospective review of DTC pts treated at a tertiary center with BRAFi ± MEKi following molecular testing for both BRAF and TERTp mutations and at least one restaging visit. RECIST v1.1 was used for best response assessment. Progression free survival (PFS) was estimated using Kaplan‐Meier.27 pts met inclusion criteria; 52% males; age at diagnosis was 57.5 ± 11 years (mean ± SD); prior tx: surgery(93%); RAI(70%); median RAI cumulative activity of 115mCi (Interquartile 0, 215); neck XRT(26%) and multikinase inhibitors(11%). 24/27(89%) pts had dual BRAFV600E and TERTp mutations (22 had ‐124C>T and 2 had ‐146C>T) and 3(11%) had only BRAFV600E mutation. Of 24 pts with dual mutations, 10 had BRAFi + MEKi combination and 14 had BRAFi monotx. The 3 pts with BRAFV600E mutation were treated with BRAFi monotx. BRAFi was dabrafenib in 25(93%) and vemurafenib in 2(7%) pts. Trametinib was the only MEKi used in combination with dabrafenib. Median follow up was 8.9 months (interquartile 4.3, 15.2). Best response in pts with dual mutations: 5/10(50%) PR and 5/10(50%) SD with combination tx; 4/14(29%) PR and 10/14(71%) SD with monotx (p = 0.2). The 3 pts with only BRAF mutation had SD on monotx. In pts with dual versus single mutation, median PFS was 25.5 months versus 4.6 months (p = 0.047). The median PFS was 25.5 months for pts who received monotx and not reached for pts who received combination tx (p = 0.66).). Drug holds were needed in 9(33%) pts (3 were on dual tx); dose reductions in 8(30%) pts (3 were on dual tx) and 2 pts discontinued dugs due to AEs. The prevalence of dual BRAFV600E and TERTp mutations in progressive DTC pts tested for both mutations prior to starting systemic tx was 89%. Coexisting mutations may be linked with a better response to targeted tx. However, the difference observed with BRAFi + MEKi versus BRAFi alone was not significant. Further studies are needed to confirm these results.

THE ADDITION OF MICRORNA EXPRESSION TESTING INCREASES THE SENSITIVITY AND SPECIFICITY TO DETECT THYROID CANCER IN PEDIATRIC PATIENTS.

A. Franco¹, S. Mostoufi‐Moab^1,2, L. Surrey³, A. Isaza¹, K. Kazahaya⁴, S. Adzick⁵, E. Labourier⁶, A.J. Bauer¹

¹Endocrinology and Diabetes, Children's Hospital of Philadelphia, Haddonfield, NJ; ²Oncology, Children's Hospital of Philadelphia, Philadelphia, PA; ³Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA; ⁴Otolaryngology, Children's Hospital of Philadelphia, Philadelphia, PA; ⁵Surgery, Children's Hospital of Philadelphia, Phildelphia, PA; ⁶Consultant, Austin, TX

Detection of thyroid mutations is associated with an increased risk of malignancy across a spectrum of predictability from highly predictive, including BRAF V600E and fusions in RET, NTRK, and ALK, to lower predictability, including RAS, PTEN, DICER1, and PAX8/PPARg. The inability to detect a mutation does not decrease the probability of malignancy. The aim of this study was to assess whether adding a 10‐microRNA classifier improves the diagnostic accuracy in pediatric patients. Descriptive, retrospective evaluation of 113 archived thyroid nodule samples (47 benign and 66 malignant); 31 classic‐PTC (cPTC), 20 fvPTC, 9 diffuse sclerosing variant‐PTC, and 6 FTCs. All slides were reviewed for adequacy of sample and accuracy of diagnosis. Molecular testing of residual total nucleic acids containing DNA, mRNA and miRNA was carried out using ThyGeNEXT^Ò and ThyraMIR^Òassays (Interpace Diagnostics, Parsippany, NJ). Genetic alterations associated with an increased risk for malignancy were detected in 35 malignant cases (53%) with 43% of these cases exhibiting a gene rearrangement, RET/PTC (n = 9), STRN/ALK (n = 2), ETV6/NTRK3 (n = 2) or PAX8/PPARG (n = 2). The miRNA signature associated with a high risk for malignancy was detected in 11 thyroid oncogene mutation‐negative malignant cases (35%). None of the benign cases were false positive with the miRNA classifier. Overall, the combination ThyGeNEXT^Ò and ThyraMIR^Òtesting resulted in a sensitivity of 70% (95% CI: 57–80%) and specificity of 96% (95% CI: 85–99%) to detect a thyroid malignancy. These data suggest that the regulatory miRNA pathways underlying thyroid tumorigenesis may be similar in adult and children. Combined testing for well‐established somatic gene alterations and miRNA gene expression improved the molecular classification of pediatric thyroid nodules. Incorporation of a miRNA expression classifier into the preoperative assessment of indeterminate cytology nodules and negative oncogene testing is predicted to improve the diagnostic accuracy for detecting malignancy as well as enhance and optimize risk stratification of thyroid surgery in pediatric patients.

MORE ACTIVE RAI THERAPY COULD NOT PROMISE BETTER RESPONSES IN BRAF^V600E MUTANT PTC PATIENTS WITH N1B DISEASE OR EXTRA‐THYROID EXTENSION

Y. Liu, X. Zhang, Y. Lin

Department of Nuclear Medicine, Peking Union Medical College Hospital, Beijing, China

BRAF ^V600E mutant papillary thyroid cancer(PTC) patients with N1b disease or extrathyroid extension(ETE) generally carry worrying prognosis. While, whether a high‐dose(100–150mCi) radioiodine(RAI) therapy could promise a better outcome than low‐dose(30mCi) RAI therapy for those patients remains controversial. In this study, we applied the response to therapy restratification system to evaluate the dynamic responses to low‐dose and high‐dose RAI therapy in BRAF ^V600E mutant PTC patients with N1b disease or ETE. A total of 265 BRAF ^V600E mutant non‐distant‐metastatic PTC patients who underwent thyroidectomy and RAI therapy were retrospectively enrolled. Responses to RAI therapy were dynamically evaluated and classified into excellent(ER), indeterminate(IDR), biochemical incomplete(BIR) and structural incomplete response(SIR). Patients were divided into subgroups according to pathological features including N1b disease, microscopic ETE and gross ETE to compare their responses to low‐dose and high‐dose RAI therapy. The clinicopathological characteristics and preablation stimulated thyroglobulin level was not statistically different between low‐dose and high‐dose groups in each pathological subgroup. Defining the first detection of ER as the endpoint, high‐dose RAI therapy was not correlated with less time to achieve ER or a higher ER curve than low‐dose RAI therapy in the N1b(4.73 vs 4.85months, p = 0.611), microscopic ETE(4.4 vs 3.0months, p = 0.130), gross ETE(3.8 vs 2.5months, p = 0.027), N1b and microscopic ETE (4.4 vs 3.1months, p = 0.402) and N1b and gross ETE(4.6 vs 2.8 months, p = 0.237) subgroups. At the end of follow‐up(median 31.8 months), high‐dose RAI therapy was not correlated with a higher ER rate than low‐dose RAI therapy in N1b(72.5% vs 73.9%, p = 0.602), microscopic ETE(69.3% vs 83.5%, p = 0.006), gross ETE(81.0% vs 100%, p = 0.195), N1b and microscopic ETE(70.6% vs 70%, p = 0.924), and N1b and gross ETE(75% vs 100%, p = 0.310) subgroups. For BRAF ^V600E mutant PTC patients with N1b disease or ETE, more active RAI therapy couldn't promise better responses than low‐dose RAI therapy. Integrating the therapeutic efficacy and radiation side effects, low‐dose RAI therapy might be a better choice for those patients.

THYROID CYTOLOGY SMEAR SLIDES: AN UNTAPPED RESOURCE FOR THYROSEQ TESTING

M.N. Nikiforova¹, M. Lepe³, L. Tolino², A.I. Wald², Z.W. Baloch³, Y.E. Nikiforov¹

¹University of Pittsburgh, Pittsburgh, PA; ²University of Pittsburgh Medical Center, Pittsburgh, PA; ³University of Pennsylvania Medical Center, Philadelphia, PA

Molecular testing (MT) of thyroid nodules diagnosed as indeterminate on fine‐needle aspiration (FNA) cytology is increasingly used to guide patient management. Although FNA samples collected into a preservative solution represent an ideal sample type, in some cases FNA smear slides are the only specimen available for MT. In this study, we evaluated the performance of ThyroSeq V3 GC in thyroid FNA smear slides.

ThyroSeq v3 GC analysis was blindly performed on 31 routinely prepared cytology smears from 16 patients with prior ThyroSeq testing on FNA samples collected directly into preservative solution. Seventeen air‐dried and Diff‐Quik^® stained smears (DQSS) and 14 alcohol‐fixed and Papanicolaou‐stained (PAP) smears were used. Each tested FNA smear contained at least 10 cell groups with 10–20 cells per group. After un‐blinding, the findings in cytology smears were compared to those obtained from the original FNA sample and to the histopathologic follow‐up.

Overall, 25/31 smears (81%) yielded adequate quantity and quality of nucleic acids for informative ThyroSeq analysis. This included 14/14 (100%) PAP and 11/17 (65%) DQSS. The 6 DQSS had poor quality of RNA and failed testing, whereas DNA was acceptable in all cases. In samples with adequate nucleic acids, the overall accuracy of detecting molecular alterations was 98% as compared to originally tested freshly collected FNA samples. This included a 100% concordance for mutations, 96% for fusions, 94% for copy number alterations, 100% for gene expression, and 100% for parathyroid cells. Testing of informative cytology smears yielded a Positive ThyroSeq test result in all nodules classified as Positive in the original samples. Surgical pathology diagnosis correlation revealed that 67% of nodules were cancers/NIFTP and 33% benign follicular adenomas. Thyroid FNA cytology smear slides with adequate cellularity can be successfully used for ThyroSeq GC testing in ∼80% of cases, with higher informative rate in PAP smears. When an adequate amount of nucleic acid is available, all types of genetic alterations can be accurately detected. Testing of routine cytology smears may eliminate the need to repeat an FNA biopsy for patients with indeterminate cytology.

DIFFERENTIATED THYROID CANCER IN OLDER ADULTS: DOES A TREATMENT DISPARITY EXIST?

W. Sutton², A. Mathur², J. Canner², M. Zeiger¹, D. Segev², M.A. Munir²

¹Surgical Oncology, National Cancer Institute, Bethesda, MD; ²Department of Surgery, Johns Hopkins University, Baltimore, MD

The growth of the aging population coupled with the increasing incidence of thyroid cancer warrants a better understanding of thyroid cancer in older adults. Our primary objective was to investigate if extent of surgery affects disease‐specific mortality in older adults with differentiated thyroid cancer. We performed a population‐based study using the Surveillance, Epidemiology, and End Results (SEER‐18) program to examine adults with differentiated thyroid cancer between 2004–2015. Patients were stratified by age: younger adults (18–64 years), older adults (65–79 years), and super‐elderly (≥80 years). Kaplan‐Meier curves were used to estimate disease‐specific mortality. Multivariable cox regression was performed to assess associations between clinico‐pathological characteristics and treatment patterns on overall and disease‐specific mortality. Of 123,842 patients with differentiated thyroid cancer, 101,296 were younger adults, 19,483 were older adults, and 3,400 were ≥80 years old. In patients with differentiated thyroid cancer, fewer older adults and super‐elderly underwent any surgery (96.6% vs. 93.1% vs. 78.9%, P: <0.001) or received RAI (46.1% vs. 38.4% vs. 27.3%, P: <0.001). Furthermore, older adults and super‐elderly had higher risks of dying from thyroid cancers (HR: 4.6, 95% CI: 4.0–5.2, P: <0.001, and HR: 10.8, 95% CI: 9.2–12.7, P < 0.001 respectively). On multivariable Cox regression, management of thyroid cancer was a significant predictor of mortality. The most common reason that patients did not undergo cancer‐directed surgery was that it was not recommended (1.61% vs. 3.23% vs 13.24% in younger adults, older adults, super‐elderly), whereas the least common reason was that it was not recommended due to patient comorbidities (0.05% vs. 0.31% vs. 1.56%). In patients with well‐differentiated thyroid cancer, fewer older adults underwent any surgery or treatment with RAI, perhaps contributing to worse survival than their younger counterparts. The most common reason that patients with differentiated thyroid cancer did not undergo cancer‐directed surgery was that it was not recommended. Future prospective studies are needed to assess this age‐related treatment disparity.

THE CORRELATION BETWEEN 131I UPTAKE AND THERAPEUTIC EFFICACY IN DISTANT METASTATIC DIFFERENTIATED THYROID CARCINOMA: A RETROSPECTIVE MULTICENTER STUDY

J. Tan¹, Z. Gao², W. Ouyang³, W. Chen⁴, C. Lou⁵, Z. Wei⁶, Y. Lin⁷, R. Wang¹, R. Zhang¹

¹Nuclear Medicine, Tianjin Medical University General Hospital, Tianjin, China; ²Nuclear Medicine, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; ³Nuclear Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China; ⁴Nuclear Medicine, Fujian Provincial Hospital of Fujian Medical University, Fuzhou, China; ⁵Nuclear Medicine, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China; ⁶Nuclear Medicine, First Affiliated Hospital of Guangxi Medical University, Nanning, China; ⁷Nuclear Medicine, Peking Union Medical College Hospital, Beijing, China

The therapeutic efficacy of radioiodine (131I) on the metastatic differentiated thyroid carcinoma (m‐DTC) is affected not only by the administrated 131I dose, but also the exact lesional 131I uptake. The purpose of this study is to evaluate the correlation between lesional 131I uptake and therapeutic efficacy in patients with m‐DTC. A retrospective multicenter study was conducted in China. All patients were confirmed as DTC, diagnosed as local or distant metastases either pathologically or suggested integrating the serum Tg/TgAb, postoperative 131I imaging, neck ultrasound, CT and/or MRI. The lesional 131I uptake capacity was quantitative analyzed as its target‐to‐nontarget ratio (T/NT) through the regions of interest. The efficacies were divided into complete remission (CR), partial remission (PR), stable disease (SD) and progress disease (PD) based on RECIST criteria. We further classified CR and PR as effectiveness, while considered SD and PD as no remission (NR). Factors including lesional T/NT, sex, age at diagnosis, stimulated Tg/TgAb, 131I dose and interval time between oral 131I and imaging, were assessed by univariate and multivariate analyses.(1)Totally, 1165 efficacies (including 138 patients, 360 lesions) were evaluated. In the planar imaging, T/NT values of CR, PR, SD and PD were 27.54 ± 49.22,15.18 ± 25.55, 13.53 ± 28.95 and 13.99 ± 12.80, respectively, with no significant differences (c2 = 4.15, P = 0.246);(2) In the tomographic imaging, the corresponding T/NT values were 37.16 ± 33.96, 33.61 ± 48.61, 22.90 ± 42.67 and 11.17 ± 15.01 respectively, with significant differences (c2 = 30.46, P < 0.001). Besides T/NT, univariate analysis also showed that stimulated Tg, 131I dose and age were the factors affecting therapeutic efficacy. Furthermore, binary logistic regression analysis revealed that patients with lower T/NT lesions (OR: 1.186, P = 0.006) and older cases (OR: 1.134, P = 0.022) had a higher probability of NR. The metastatic lesions with different 131I uptake capacities in the postoperative 131I SPECT/CT imaging showed different responses to 131I therapy. Our study confirmed that older patients with lower T/NT metastatic lesions usually have poor therapeutic efficacy.

INFLAMMATORY STIMULUS DECREASES MYOGENESIS DURING NONTHYROIDAL ILLNESS SYNDROME

F.F. Bloise¹, T.S. Oliveira¹, C.B. Andrade¹, K.F. Novaes¹, N. Blanco¹, P.L. Silva¹, P.R. Rocco¹, S. Wajner², A.L. Maia², T.M. Ortiga‐Carvalho¹

¹Instituto de Biofisica Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio De Janeiro, Brazil; ²Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

Sepsis can cause nonthyroidal illness syndrome (NTIS), an unbalance in thyroid hormones (TH) signaling. Previously, we demonstrated that local TH signaling is decreases in diaphragm during acute bacterial sepsis. Septic patients usually present respiratory failure associated with diaphragmatic dysfunction, which is the mains respiratory muscle. Knowing that T3 is an important regulator of muscle function and its signaling is modulated by sepsis, our hypothesis is that sepsis‐induced NTIS is associates with impaired diaphragm funcion. Chronic sepsis model was induced by cecal ligation and puncture (CLP). CLP or sham surgery was performed in 12 weeks‐old C57Bl/6 male mice (9 animals per group). All animals received antibiotic (imipenem 25mg/kg) daily starting 8 hours after surgery. Diaphragm was collected 120h after the surgery. Data are expressed as mean ± SEM. We investigated mRNA expression by qPCR and morphology by electron microscopy. Cell culture experiment was performed using myoblast C2C12 stimulated or not (CTR) with 10ng/ml LPS for 24h. Chronicle sepsis induced NTIS (liver Dio1 expression decreased by 37% and Dio3 increased by 3 times). The diaphragm Thra and Dio2 expression did not change. Morphological analysis suggested an increase in tissue degeneration of the CLP diaphragm. This was associated with a decrease in Myh7 (0.99 ± 0.29 sham, 0.58 ± 0.31 CLP) and Myh4 expression in the diaphragm (1.0 ± 0.4 sham, 0.38 ± 0.23 CLP), which suggests a decrease in skeletal muscle regeneration. To better understand the impact of NTIS in skeletal muscle regeneration (myogenesis) we performed an in vitro experiment. We observed a decrease in Thra (77%) and an increase in D2 activity (double) in LPS group. The myogenic marker (Myod1) were decrease by 75% in LPS compared to CTR. In conclusion, our in vivo and in vitro data suggest that inflammatory stimulus decreases myogenesis, which could be related to NTIS.

THYROID FUNCTION TESTS IN PATIENTS WITH POSTURAL ORTHOSTATIC TACHYCARDIA SYNDROME AND TEMPERATURE INTOLERANCE

S.A. Shaheen, E. Hasbay, E. Hasbay, C. Peralta, M. Abdeen, H. Fatima, A. Suleman

Cardiology, The Heartbeat Clinic, McKinney, TX

Postural orthostatic tachycardia syndrome (POTS) is a common form of autonomic dysregulation characterized as an excessive tachycardia upon standing in the presence of orthostatic intolerance. Current adult diagnostic criterion requires a heart rate increase of greater than or equal to 30 bpm within the initial 10 minutes of standing or head‐up tilt in the absence of orthostatic hypotension.

Thyroid Hormones (TH) play a significant role in energy expenditure through both central and peripheral actions. TH maintains basal metabolic rate, facilitates adaptive thermogenesis, modulates appetite and food intake, and regulates body weight.

The aim of the study is to look for any abnormality/change in thyroid function tests in POTS patients having temperature intolerance in the absence of thyroid disease.

A retrospective study was conducted by analyzing Electronic Medical Records of randomly selected 141 POTS patients who were not diagnosed with any thyroid disorder. Their thyroid function tests including TSH, FT3, FT4 were reviewed. Patients having heat intolerance (HI) and cold intolerance (CI) were also identified.

141 POTS patients (Females = 135, Males = 6) with mean age 32 yrs ±12. Out of 141 patients 86 (61%) had CI and 93 (66%) had HI. The mean of TSH, FT3, FT4 in patients having CI is TSH 2.17 ± 1.32, FT3 3.67 ± 2.48, FT4 1.48 ± 1.69 as compared to the patients without CI, TSH 1.87 ± 1.04, FT3 3.55 ± 2.48, FT4 1.69 ± 1.64.

The mean of TSH, FT3, FT4 in patients having HI is TSH 2.06 ± 1.21, FT3 3.88 ± 4.35, FT4 1.49 ± 1.69 as compared to the patient without HI, TSH 2.08 ± 1.32, FT3 3.07 ± 0.66, FT4 1.69 ± 1.67.

POTS Patients having temperature intolerance are sensitive to minor changes in thyroid function tests. The POTS patients with CI & HI had slightly higher and lower levels of TSH respectively as compared to ones without HI/CI in the absence of any diagnosed thyroid disorder.

SERUM TRF‐PRO‐AGG‐018 AS A NOVEL DIAGNOSTIC BIOMARKER FOR DISCRIMINATING PAPILLARY THYROID CARCINOMA FROM NODULAR GOITER

J. Tang¹, B. Zhang²

¹Department of Ultrasound, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ²Department of Medical Ultrasonics, China‐Japan Friendship Hospital, Be, China

Context: tRNA‐derived fragments (tRFs) are emerging as a new class of small non‐coding RNAs due to their potential roles as novel biomarkers and therapeutic targets in cancer. However, their involvement in papillary thyroid carcinoma is still unclear.

Objetives: We investigated the role of tRF circulating in the blood to discriminate papillary thyroid carcinomas (PTCs) from nodular goiters (NGs).

Setting and Design: tRFs expression profile was measured in pooled sera from 6 healthy subjects, 6 nodular goiters(NG) and 6 patients with papillary thyroid cancer(PTC) using high‐throughput sequencing (cohort 1). Three tRF candidates were validated in individual samples (cohort 1) by qRT‐PCR. The confirmed tRF was further validated in a larger second cohort.

Results: Our sequence results showed that circulating tRFs were differentially expressed in patients with PTC compared with NG and HS. After the validation in individual samples, tRF‐PRO‐AGG‐018 was confirmed as differentially expressed. The specific tRF was further validated in a second cohort of sera from 42 PTC, 32NG and 32HS. Moreover, tRF‐PRO‐AGG‐018 yielded high diagnostic accuracy in discriminating PTC from NG.

Conclusions: We found that tRF‐PRO‐AGG‐018 could accurately discriminate papillary thyroid carcinoma from thyroid nodules, which may have considerable clinical value in differential diagnosis of thyroid nodules.

UTILITY OF THE TARGETED NEXT‐GENERATION SEQUENCING PANEL (THYROSEQ) FOR THE MANAGEMENT OF THYROID NODULES IN A PRIVATE PRACTICE SETTING

O. Anochie, D. Mehta, A. Seth, A. Okoh

Internal Medicine, Newark Beth Israel Medical Center, Newark, NJ

Background: The ThyroSeq mutational panel gene expression classifier (GEC) is used to risk stratify cytologically indeterminate thyroid nodules. In the current study, the performance of this test was evaluated in a real‐world private practice setting.

Materials and Methods: A prospectively maintained thyroid database at a private practice was retrospectively reviewed to identify indeterminate nodules that were managed by using the ThyroSeq GEC testing. For nodules that were surgically resected, surgical pathology findings were studied within the context of molecular testing findings, comparing performance stratified by the American Thyroid Association (ATA 2015) guidelines. Total of 492 thyroid nodules were evaluated in 13 months (December 2017 to January 2019). Thyroid FNA reports showed benign findings in 440 (89%), atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS) in 45(9%) and follicular neoplasm or suspicious for a follicular neoplasm or malignancy in 7(1.4%). Of the 45 nodules that were evaluated with Thyroseq, 32 were reported as low risk for malignancy. The mutations reported were TSHR, PTEN and EZH1. 12 were intermediate to high risk with mutations MEN1, DICER1, NRAS, KRAS, gene expression profile, EIFIAX, cope number mutations. 6 of 12 patients who underwent hemi/total thyroidectomy had benign surgical pathology. One patient had PTC, follicular variant, 4 cm and another had follicular thyroid cancer. Interestingly, both tested positive for gene expression profile, one for NRAS. Unfortunately, we had 4 patients who were lost to follow up, two with high risk Thyroseq with MEN1 and DICER1 mutation.

Results show that most Bethesda Category III/IV nodules with Thyroseq showing intermediate to high risk probability resulted in benign surgical pathology. A future study with a much larger sample size would be interesting to investigate the sensitivity of Thyroseq results/specific mutations in predicting malignant potential after review of pathology specimens in a private practice setting.

POSITIVE PREDICTIVE VALUE OF NTRK, RET, BRAF, AND ALK FUSIONS IN BETHESDA III/IV THYROID FINE‐NEEDLE ASPIRATES

B.C. Stack¹, P. Sadow², M.I. Hu³, M.J. Livhits⁴, S.I. Sherman³, S. Ali⁵, J.F. Krane⁶, D.B. Evans⁷, Y. Hao⁸, J.E. Babiarz⁸, G.C. Kennedy^8,9, R.T. Kloos⁹

¹Department of Otolaryngology‐Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, AR; ²Pathology Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA; ³Department of Endocrine Neoplasia and Hormonal Disorders, University of Texas M. D. Anderson Cancer Center, Houston, TX; ⁴Department of Surgery, Section of Endocrine Surgery, Ronald Reagan UCLA Medical Center, Santa Monica, CA; ⁵Departments of Pathology and Radiology, Johns Hopkins University School of Medicine, Baltimore, MD; ⁶Department of Pathology, David Geffen School of Medicine at UCLA, Los Angeles, CA; ⁷Department of Surgery, The Medical College of Wisconsin, Milwaukee, WI; ⁸Research and Development, Veracyte, Inc., South San Francisco, CA; ⁹Medical Affairs, Veracyte, Inc., South San Francisco, CA

The prevalence of thyroid cancer, as determined by excision, among biopsied Bethesda III/IV nodules with fusions of ALK, BRAF, NTRK or RET (other than RET/PTC1 and RET/PTC3), is unknown. The Afirma Genomic Sequencing Classifier and Xpression Atlas report 130 fusion pairs, including these fusions. Here we report their PPVs in real‐world clinical practice. Consecutive cohorts of Bethesda III/IV nodules with ALK, BRAF, NTRK or RET fusions (other than RET/PTC1 and RET/PTC3) submitted to Veracyte for molecular analysis were identified. Local surgical pathology diagnoses were sought with IRB approval. Only one nodule per patient was included. PPV calculations did not consider NIFTP as malignant. Gene pairs are listed alphabetically. Local surgical pathology diagnoses were available for 58 thyroid nodules from 58 patients. No sample had a concurrent variant or second fusion. Twelve ALK fusion partners included 8 STRN and 4 EML4. Eight (67%) were malignant while 4 were adenomatoid or hyperplastic (3 STRN and 1 EML4). Sixteen BRAF fusion partners included 5 AGK, 5 SND1, 2 CCNY, 2 MKRN1, 1 POR, and 1 MACF1; 12 carcinomas (75%), 1 NIFTP (AGK), and 3 adenomas (1 each AGK, SND1, MKRN1). Twenty‐three NTRK fusion partners included 19 ETV6, 2 TPM3, and 2 RBPMS; 22 carcinomas (96%) and 1 hyperplastic nodule. Seven RET fusion partners included 3 ERC1, 1 TRIM33, 1 AKAP13, 1 PRKAR1A and 1 FKBP15; 6 carcinomas (86%) and 1 NIFTP (ERC1).NTRK and RET fusions among Bethesda III/IV nodules were associated with malignancy in 28 of 30 nodules. Risk of malignancy was lower among nodules with ALK (67%) or BRAF (75%) fusions. We found it notable that two nodules with fusions expected to be BRAFV600E‐like were diagnosed as NIFTP. Additionally, 4 nodules were reported as hyperplastic despite harboring fusions expected to drive neoplasia (2 ALK/STRN, 1 ALK/EML4, 1 ETV6/NTRK3). With a modest sample size, our findings highlight the local histopathology risk of malignancy associated with several fusions among nodules with indeterminate cytopathology. Future studies with expert histopathologists may provide additional comparative insight, as will long term clinical outcomes associating fusion partners with biological risk for recurrence.

HASHIMOTO'S DISEASE RESEARCH HAS HISTORICALLY BEEN UNDERREPRESENTED AT THE AMERICAN THYROID ASSOCIATION MEETINGS 2015–2019

B. Henderson, E. Pilgrim, J. Henderson

Charleston Thyroid Center, Mount Pleasant, SC

Hashimoto's Disease affects more than 14 million Americans¹, making it the most common form of thyroid illness in the United States. Some estimate that millions more have Hashimoto's Disease but remain undiagnosed. Despite this, there is a paucity of clinical research on hypothyroidism and Hashimoto's Disease, in particular. All lectures presented at the American Thyroid Association meetings from 2015–2019 were identified and classified into one of the following six categories: thyroid cancer, hypothyroidism, Hashimoto's Disease, hyperthyroidism/Graves' Disease, thyroid hormone action, and other. Descriptive and statistical analysis was performed. From 2015–2019, only 5% of lectures were dedicated to Hashimoto's Disease research. An additional 6% of lectures discussed hypothyroidism. In the past 5 years, an overwhelming majority of lectures at the ATA annual meetings have been dedicated to thyroid cancer research (45%), followed by thyroid hormone action (19%), hyperthyroidism/Graves' Disease (10%), and other (15%). The 2019 ATA meeting has put more emphasis on hypothyroidism than previous years, with 14.3% of lectures on hypothyroidism (compared to 6% historically on average), whereas Hashimoto's Disease remains at 5.1% of total lectures (historically 4.1–6.4% of lectures). An overwhelming majority of lectures at the ATA meetings 2015–2019 have been dedicated to thyroid cancer research (45% of lectures) while Hashimoto's Disease Research has been categorically underrepresented (5% of lectures) (p < 0.05). Currently there are over 14 million Americans living with Hashimoto's Disease while thyroid cancer affects a little over 800,000.²

DIGITAL TOOLS MAY HELP PHYSICIANS OPTIMIZE PATIENT SYMPTOMS AND INDIVIDUALIZE TSH TARGETS

V. Högqvist Tabor¹, B. Henderson², M. Högqvist Tabor¹

¹Boost Thyroid by VLM Health, Berlin, Germany; ²Charleston Thyroid Center, Mount Pleasant, SC

Current normal TSH ranges do not adjust for age, ethnicity, time of day, and other potential confounding variables. TSH treatment goals typically vary from practitioner to practitioner and are wide in both range and variability. In clinical practice patients experience symptoms that correlate to their TSH level, even when TSH is considered in the normal range. Evaluating how TSH correlates with a longitudinal patient record of symptoms, medication dosing adjustments, and supplements may help establish optimal TSH goals for individual patients.

BOOST Thyroid is a science‐based mHealth app built on academic research on autoimmune and thyroid conditions enabling people with an underactive thyroid to log their symptoms, lab values and daily medication adherence, thereby collecting individual patient data between medical visits. Symptom data is collected on a 5‐point intensity scale from 1 (poor) to 5 (symptom‐free state). BOOST Thyroid app has over 30,000 downloads to date, and is working on analyzing over 1 million anonymized data points.

In order to determine if there is a “golden standard” TSH value that can be applied across the very diverse patient population, we have compared symptoms of people within 4 distinct TSH ranges (<0.5mIU/L; 0.5–2.0mIU/L; 2.0–5.0mIU/L and 5–10mIU/L), and have observed five main symptoms patients have tracked: headache, energy, sleep quality, focus and digestion. We observed minimal difference in the average symptom complaints between distinct TSH level groups for the sleep quality, ranging from poor (value 1) in the group of patients TSH under 0.5 mIU/L to average (value 3) in the other three groups. The other four symptoms did not differ between the groups. Moreover all the symptoms in each group ranged from value 1 and 5, demonstrating widespread symptom diversity. At the ATA meeting, we will further report on distinct cohorts of patients based on the combination of the TSH and symptom data, as well as T4 values, where data is available.

In summary, digital tools can facilitate patients and doctors conversation, and create an opportunity for a true shared decision making which can lead to health improvements, reduction of secondary problems, and optimal disease management.

THYROTROPIN STIMULATION OF THYMIC STROMAL LYMPHOPOIETIN PRODUCTION BY HUMAN ADIPOCYTES

L. Ma, J. Zhen, A. Sorisky

Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

Thymic stromal lymphopoietin (TSLP) is an inflammatory cytokine recently recognized to be expressed in human adipocytes stimulated by thyrotropin (TSH). This study aimed to identify TSH‐dependent signaling routes that up‐regulate TSLP and to examine if TSLP production is adipose depot‐dependent. Abdominal subcutaneous adipose tissue was obtained from 9 female patients undergoing elective surgery (approved by the Ottawa Health Science Network Research Ethics Board, #1995023‐01H). Human abdominal differentiated adipocytes were stimulated with TSH. Activation of signal transduction pathways was measured by immunoblotting, and TSLP levels in cell culture medium were assessed by ELISA. TSLP responses from abdominal subcutaneous and omental adipocytes were compared. TSH‐stimulated TSLP secretion from human abdominal subcutaneous differentiated adipocytes was enhanced by IBMX (raises cAMP levels) and was blocked by UO126 (inhibitor of MEK1/2‐ ERK1/2 pathway). No inhibition was observed with SB203580 (p38 MAPK inhibitor) nor SP600125 (JNK inhibitor). Interferon‐gamma, but not interleukin‐4, inhibited TSLP responses to TSH. Intra‐abdominal omental adipocytes also released TSLP in response to TSH. TSLP is produced by human differentiated adipocytes derived from subcutaneous or omental depots in response to TSH. Our data suggest a role for PKA and the MEK1/2‐ERK1/2 pathway in mediating the TSH effect on TSLP. TSLP may be of relevance to the extra‐thyroidal action of TSH associated with adipose dysfunction and the cardiovascular disease risk observed with the elevated TSH levels of subclinical hypothyroidism.

WIDE‐RANGING AND UNMEASURED RESIDUAL THYROID FUNCTION OBSCURES ASSESSMENT OF COMBINATION LT4+LT3 THERAPY SUCCESS

J. DiStefano¹, J. Jonklaas²

¹Computer Science & Medicine, UCLA, Los Angeles, CA; ²Georgetown University, Washington, DC

Introduction: Results and success of 2 decades of combination therapy studies have been mixed. We hypothesized that residual thyroid function (RTF), which can be quite variable in hypothyroid patients, especially those with different etiologies, might explain the differences.

Methods: We defined trial success as improved clinical outcome measures and/or patient preference for added LT3, with success depending strongly on RTF as well as LT3 added to sufficient LT4 dosing to normalize serum T4 and TSH, all rendering T3 levels to at least mid‐normal range. We used the THYROSIM app to simulate ‘‘what‐if’’ experiments in patients and study designs corresponding to 13 published study trials. The app graphically provided serum total T4 and free (FT4), total T3 and free (FT3), and TSH responses over time, to different simulated LT4 and combination LT4+LT3 dosage inputs in patients with primary hypothyroidism. We compared simulation results with available study response data, computed RTF values that matched the data, classified and compared them with trial success measures.

Results: Simulation results generated 3 categories of patients with different RTFs and T3 & T4 levels at trial endpoints. Four trial groups had >20%, four <10%, and five 10–20% RTF. Four trials were predicted to achieve high, seven medium, & two low T3 levels. We were able to correctly predict 12 of 13 trials deemed successful or not. We then generated an algorithm for optimizing dosage combinations suitable for different RTF categories, with the goal of achieving mid‐range‐normal T4, T3 and TSH levels. RTF is estimated from TSH, T4 or T3 measurements prior to any hormone therapy treatment, using 3 new nomograms for computing RTFs from these measurements. Recommended once‐daily starting doses are:100μg LT4 + 10–12.5μg LT3; 100μg LT4 + 7.5–10μg LT3; & 87.5 μg LT4 + 7.5 μg LT3; for <10%, 10–20%, & >20% RTF, respectively.

Conclusion: Variable RTF is a complicating factor in assessing success of combination LT4+T3 therapy. We have estimated and partially validated RTFs for most existing trial data, and provided an algorithm for estimating RTF from accessible data, & optimizing LT4+LT3 combination patient dosing for future combination therapy trials.

TRβ ATTENUATES PI3K SIGNALING IN ANAPLASTIC THYROID CANCER THROUGH NOVEL GENOMIC MECHANISMS TO ENHANCE PI3K INHIBITOR EFFICACY

C. Davidson, E.L. Bolf, N.E. Gillis, J. Tomczak, F. Carr

University of Vermont, South Burlington, VT

Thyroid cancer is the most common endocrine malignancy, and the incidence has tripled in the past few decades. Papillary and follicular thyroid carcinomas are well differentiated, which functionally and morphologically resemble the noncancerous thyrocyte. However, anaplastic thyroid cancer (ATC) is much less differentiated, and is almost always lethal. No effective long‐term treatment options exist for ATC, as resistance to common chemotherapeutics develops. One notable feature of anaplastic thyroid carcinoma is the loss of expression of thyroid hormone receptor β (TRβ). TRβ binds thyroid hormone (T3) and regulates a variety of cellular processes, including metabolism, growth, and development, as well as suppressing tumorigenesis. TRβ or an empty vector control were transduced into the ATC cell line SW1736. The cells were treated for T3 or vehicle for 24 hours and submitted for RNA‐sequencing. PI3K signaling genes that were significantly differentially expressed were validated by qPCR using the same cell treatment conditions. T3‐treated transduced SW1736 cells were also subjugated to 50 μM LY294002 or vehicle for analysis of phosphorylated and total Akt and mTOR. To investigate the effects of TRβ and LY294002 on proliferation, transduced SW1736 cells were treated with LY294002 and lifted and counted every 24 hours. Cells were also grown to confluency, scratched with a pipet tip, and imaged every 24 hours to measure migration. SW1736 cells exhibit resistance to the PI3K inhibitor LY294002 compared to normal like thyroid cells as shown by western blot after 50 μM LY294002 treatment. However, when TRβ is transduced into the cell, PI3K signaling is attenuated as shown by reduced pAkt and pmTOR signal, and this effect is amplified in the presence of LY294002. This effect is also seen in the cell counting and scratch assays; SW1736 cells transduced with TRβ and treated with LY294002 exhibit the lowest rates of proliferation and migration. qPCR results revealed that liganded TRβ increased the expression of the phosphatases PTEN, INPP4B, INPP5J, and PHLLP1. Taken together, this work demonstrates a novel tumor suppressive function of TRβ in ATC which provides for an improved response to the PI3K inhibitor LY294002.

QUANTIFICATION OF R‐LOOP EXPRESSION IN ANAPLASTIC THYROID CARCINOMA USING AUTOMATED CELL SEGMENTATION SOFTWARE

P.C. Ramesh¹, Q. Xu⁴, P. Kilgore², U. Cvek², A. Adewoye³, P. Weinberger⁴

¹Biological Sciences, Louisiana State University at Shreveport, Shreveport, LA; ²Computer Sciences, Louisiana State University at Shreveport, Shreveport, LA; ³Department of Chemistry, Louisiana State University at Shreveport, Shreveport, LA; ⁴Otolaryngology, Louisiana State University Health Sciences Center ‐ Shreveport, Shreveport, LA

Anaplastic Thyroid Cancer (ATC) is characterized by rapid proliferation and resistance to treatment. R‐loops are 3‐strand RNA: DNA: DNA structures created when RNA polymerase collides with a replication fork. The role of r‐loops in ATC pathophysiology is poorly understood, but evidence suggests that rapid processing of r‐loops may play a role in ATC cell survival. Quantification of r‐loop signal is labor intensive, requiring measurement within non‐nucleolar nuclear regions (nNNR). InForm image analysis software allows unsupervised machine learning based on user‐defined training sets to automatically identify morphological regions of tissues. Our hypothesis was that inForm analysis could be extended to identify subcellular features such as non‐nucleolar nucleus, supporting measurement of r‐loop expression in ATC cell lines. We used standard immunofluorescence based on mouse S9.6 monoclonal antibody to identify r‐loops in ATC cell lines ACT‐1, THJ11T, and THJ16T. Multiple images were captured using water‐immersion 60x apochromatic lens. A gold‐standard of each image was prepared by manual delineation of non‐nucleolar nuclear regions (nNNR), and computer algorithms for each cell line were generated using training regions ranging from one cell per image to 8 cells per image then applied to all cells. For all conditions studied, inForm successfully delineated nNNR regions with high accuracy, sensitivity, and specificity: ACT1 accuracy 91%, sensitivity 84%, specificity 95%; THJ11T accuracy 96%, sensitivity 84%, specificity 98%; THJ16T accuracy 91%, sensitivity 89%, specificity 91%. A clear trend was observed: by the 8‐cell experiment, sensitivity was maximal (ACT‐1 92%, THJ11T 84%, THJ16T 87%) while specificity was minimal (ACT‐1 92%, THJ11T 99%, THJ16T 94%). There was significantly more time required for the manual gold‐standard designation compared to the AI workflow (including training and designation) at the 1‐cell (11.7 +/‐ 1 min v. 7.1 +/‐5 min), 2‐cell (21.6 +/‐ 2 min v. 12.4 +/‐10 min), 4‐cell (42.6 +/‐ 3 min v. 23.3 +/‐20 min), and 8‐cell (86.1 +/‐4 min v. 45.9 +/‐ 41 min), p = 0.09, 0.00005, 0.00008, and 0.0004 respectively.

ULTRASOUND (US)‐GUIDED PERCUTANEOUS LASER ABLATION (PLA) OF THE THYROID GLAND WITH COMPUTER TOMOGRAPHY (CT) AND HISTOPATHOLOGY CORRELATION: A SWINE MODEL FOR OPTIMIZATION OF A LOCALIZED THERAPY FOR MICROPAPILLARY THYROID CANCER (MPTC)

J.C. Camacho, F. Ridouani, S. Solomon, D. Li, R. Wong, J. Fagin, S. Monette, R. Tuttle

Memorial Sloan Kettering Cancer Center, New York, NY

Evaluate the safety and feasibility of US‐guided PLA in a swine model and establish optimal energy parameters for localized therapy in mPTC.IACUC approved study in a prospective cohort of swine that underwent US‐guided PLA of normal thyroid tissue (Elesta Echolaser X4 with Orblaze technology, 1064nm). Contrast‐enhanced CT (CECT) was used to assess the ablation zone size. Hydrodissection with saline was performed to protect adjacent structures. Using US, a 21‐gauge needle was advanced to the periphery of the thyroid and the laser fiber was advanced through the needle into the gland. Two cohorts were established to provide parameters to treat 1 cm lesions with a 2 mm oncologic margin (n = 5 at 3W/1400J and n = 5 at 3W/1800J). Euthanasia was performed 48 hrs following ablation with histological evaluation of the thyroid tissue performed in 4 animals. In a total of 10 swine, no procedural complications were encountered. No evidence of damage to surrounding structures was identified on CECT, during post‐procedure observation or necropsy. The mean ablation volume measured on gross pathology was consistently and significantly larger in the 3W/1800J group than in the 3W/1400J group (1.13 ± 0.13 mL vs. 0.58 ± 0.05 mL, respectively, p = 0.011). Assessment of the volume of ablated tissue on CT was strongly correlated with the volume measurements on macroscopic pathology (r = 0.98, p = 0.00017). Histological examination confirmed coagulation necrosis in the ablation zone. PLA of the thyroid is safe and feasible in a swine model. The size of the ablation zone in thyroid tissue is predictable, reproducible and correlates with the applied energy parameters. The data provided the necessary information to proceed with implementation of a clinical trial utilizing the Elesta Echolaser X4 with Orblaze technology and the Elesta ESI‐Echolaser Smart Interface as a planning support tool to histologically verify complete tumor ablation in a cohort of patients with mPTC.

TOPOISOMERASE IIα IS IMPLICATED IN ANTIPROLIFERATIVE EFFICACY OF YM155 IN ANAPLASTIC THYROID CANCER

Q. Xu¹, R. Mackay¹, A. Adewoye², P. Weinberger¹

¹Otolaryngology, lsuhsc Shreveport, Shreveport, LA; ²Public Health, Louisiana State University Shreveport, Shreveport, LA

Anaplastic thyroid cancer (ATC) is highly aggressive and resistant to traditional chemotherapeutic agents and novel treatment options are desperately needed. YM155 is an orphan drug first screened as a survivin inhibitor but abandoned after unimpressive phase II studies showed no association between response and survivin expression. Surprisingly, it was the most effective compound in a high‐throughput screen of 3,282 drugs against a panel of ATC cell lines. We hypothesized the mechanism of action for YM155 in ATC involves additional targets either in addition to (or instead of) survivin. In vitro data were obtained from three human ATC xenograft cell lines and human primary benign thyroid cells (pBTC) from surgical specimens. RNA expression (by microarray) was obtained from 16 ATC, 17 BTC, and 12 papillary thyroid cancer patient surgical specimens. We measured proliferation by alamar‐blue assay, gene expression by RT‐PCR and protein expression by capillary immune‐electrophoresis, double‐stranded DNA (dsDNA) breaks by phosphorylation of histone H2AX, and cell cycle by flow cytometry.TOP2A was overexpressed in ATC compared to PTC and pBTC patient samples (p < 0.001 for both). In ATC cell lines, Top2α was overexpressed at the gene and protein level in ACT1 and THJ16T, slightly overexpressed in ATC cell lines THJ11T and THJ29T and undetectable in PBTC. YM155 inhibited proliferation in all ATC cell lines tested with no effect on PBTC, and this anti‐proliferative activity correlated strongly with Top2α expression. YM155 reduced Top2α expression, induced DNA damage in those cells over‐expressing Top2α and caused cells to arrest in S/G2 phase. TOP2A KD by itself resulted in a modest inhibition of proliferation in ATC cells but when combined with YM155 was strongly synergistic.Top2a surprisingly plays an important role in the anti‐proliferative effect of YM155 on anaplastic thyroid carcinoma. The mechanism is unlikely to involve generation of Top2α‐DNA adducts leading to dsDNA breaks (as with Top2α‐poisons such as doxorubixin) since in the latter scenario TOP2A knockdown would be expected to reduce, not enhance drug effect. YM155 effect on ATC cells may represent a new therapeutic approach for the treatment of ATC.

IN‐SILICO MOLECULAR DOCKING REVEALS NOVEL MECHANISM OF ACTION FOR YM155, AN ORPHAN DRUG WITH STRONG PRECLINICAL EFFICACY IN ANAPLASTIC THYROID CANCER

A. Adewoye¹, Q. Xu², R. Mackay², C. Stratton³, P.C. Ramesh⁴, P. Weinberger², E. Mahdavian⁴

¹Public Health, Louisiana State University Shreveport, Shreveport, LA; ²Louisiana State University Health Science center, Shreveport, LA; ³Chemistry, Louisiana State University Shreveport, Shreveport, LA; ⁴Biological Sciences, Louisiana State University Shreveport, Shreveport, LA

YM155 is an orphan chemotherapy drug with demonstrated activity as a survivin suppressant, however efficacy in phase II trials was poor and showed no correlation with survivin expression. A recent high‐throughput screening study by the NCI surprisingly identified YM155 as having strong efficacy against ATC both in‐vitro and in‐vivo. Based on our previous study showing YM155 inhibited topologic enzymatic function of human topoisomerase II‐alpha (Top2a) in cell‐free experiments, we set out to perform molecular docking in‐silico to identify potential YM155‐Top2a interaction sites. High‐resolution crystal structure of human Top2a (1ZXM) complexed with AMPPNP was retrieved from the Protein Data Bank (PDB) database. Using AutoDock Vina 4.2 software, the crystal structure was refined including removal of heteroatoms and water molecules and assignment of Gasteiger charges. We chose as control ligands the known Top2a poison doxorubicin and known top2a catalytic inhibitors Daurinol and Salvicine. Control ligand and YM155 3D geometries were obtained from the PubChem Open Chemistry Database and converted to PDB format in Spartan. Docking was performed in AutoDock Vina including Top2a dimer, magnesium, water, and each ligand individually, with subsequent visualization using Pymol. As expected, the control ligands Daurinol and Salvicine interacted with Top2a at the ATP‐binding pocket with ‐7.2 to ‐9.8 Kcal/mol binding energies, while doxorubicin had no predicted interaction in that region but did interact at the DNA‐binding region with ‐9.8 Kcal/mol binding energy. Surprisingly, YM155 also strongly interacted with the Top2a ATP‐binding pocket with binding energies ‐8.2 to ‐8.8 Kcal/mol. Molecular Operating Environment (MOE) software allowed delineation of amino acids participating in this interaction via hydrogen bonds and hydrophobic interactions as well as significant interaction with magnesium at the binding site.

Conclusion: Our molecular docking study provides strong in‐silico data suggesting YM155 binds to the ATP‐binding pocket of human Top2a. Based on these data, we suggest that YM155 acts as a Top2a catalytic inhibitor possibly by preventing ATP hydrolysis. Further in‐vitro biochemical experiments are planned.

AMNIOTIC MEMBRANE APPLICATION TO RECURRENT LARYNGEAL NERVES APPEARS TO REDUCE TRANSIENT NERVE INJURY IN THYROID SURGERY

G.L. Clayman^1,3, R. Roy¹, J. Norman²

¹Clayman Thyroid Center, The Thyroid and Parathyroid Institute of Tampa General Hospital, Tampa, FL; ²Norman Parathyroid Center, Tampa, FL; ³Head and Neck Surgery, The University of Texas M D Anderson Cancer Center, Houston, TX

Human amnion/chorion membrane (HACM) contains growth factors, stem cells, and anti‐inflammatory effects. Our goal was to determine if HACM application on recurrent laryngeal nerves (RLN) could impact RLN injuries. Consecutive consenting patients undergoing thyroidectomy with at least a unilateral comprehensive dissection of ≥3cm of recurrent laryngeal nerve received HACM allografts. HACM patients were compared to a Control group, matched 2:1 based upon gender, age, pathology, extent of dissection and central compartment gross node involvement. Voice quality and function was self‐assessed by patients and laryngoscopy. From June 5 to July 31, 2019, sixty seven patients (56 women and 11 men) consented to the use of HACM during their thyroid operation. These 67 patients had 100 nerves (at risk) which met the criteria of a comprehensive dissection. Median patient age was 52 years (HACM, range 21–79) and 50 years (Controls, range 20–85) with females comprising 84% of both groups (all p = NS). There were 205 at risk nerves among the 134 controls. Thyroid malignancies were present in 31/67 (46%) of HACM patients with 18/31 (58%) having gross malignant involvement of central compartment, whereas the Control group had 69/134 (51%) with malignancies and 30/69 (43%) with gross central compartment disease (both, p = NS). Vocal cord injury was noted both subjectively and objectively in 1 patient (1%) in the HACM group which resolved two weeks following his operation. In contrast, the Control group had 17/134 (13%) patients (8% dissected nerves) reporting subjective or objective changes of voice decreasing to 10/134 (7.5%) at 1 week and 7/134 (5%) at 3 weeks (all p < 0.001). At one month, fiberoptic vocal dysfunction was present in 0% HACM and 5% Control (p < 0.001). All vocal dysfunction patients exhibited complete recovery within 3 months. HACM application appears to significantly reduce the incidence and duration of transient RLN injury. Our control group's laryngeal dysfunction is consistent with neuropraxic injury, due to inflammation, edema, and vascular compromise, all known targets of the biologically active HACM. A multicenter investigation of HACM for the protection of dissected RLNs during thyroid surgery is warranted.

LIMITATIONS OF DETECTING PATHOGENIC VARIANTS FROM RNA‐SEQ EXPRESSION DATA IN FNA AND TISSUE SAMPLES

C. Kaya², P. Dorsaint³, A. Sboner³, S. Mercurio², K. Wha Eng³, M.N. Nikiforova¹, O. Elemento³

¹University of Pittsburgh, Pittsburgh, PA; ²Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA; ³Weill Cornell Medicine, New York, NY

Detection of pathogenic genetic variants in resected tumor tissue of fine‐needle aspiration (FNA) samples is important for pre‐operative diagnosis or selection of therapy in thyroid and other cancer types. Although mutations occur in DNA and are typically detected using DNA sequencing, recent attempts have focused on detecting expressed variants from RNA. The aim of this study was to determine the completeness of detecting mutations from RNA as compared to DNA isolated from tumor tissue and FNA samples. DNA and RNA from 35 tissue samples were studied by whole exome sequencing (WES) and RNA‐Seq analysis at two study sites (n = 17 and n = 18). In addition, DNA and RNA from 44 thyroid FNA and 47 thyroid tissues were studied using targeted NGS 112‐gene panel and RNA‐Seq. Detection of mutations in WES and RNA‐Seq data was performed by Genome Analysis Toolkit. The mutations were filtered and annotated using ANNOVAR. Total of 162 variants was identified by WES in 35 tissue samples, of which 77 (48%) was detected by RNA‐Seq, with a detection rate of 49% by Site 1 and 46% by Site 2. Targeted NGS identified 118 pathogenic variants, of which 57 (48%) were detected by RNA‐Seq. The detection rate of variants present at >10% allele frequency (AF) was 62%, but for variants at low frequency (5–10% AF) was only 7%. The detection rate of mutations in common oncogenes (BRAF, RAS) was 94% when they were present at >10% AF in DNA; it dropped to 11% when BRAF and RAS mutations were at 5–10% AF. Tumor suppressor genes (TP53, PTEN) were detected in RNA‐Seq data at a 68% rate, and mutations in the promoter region of TERT were not detected (0%). In this study, RNA‐Seq analysis detected only 46–49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNA‐Seq was more successful for mutations occurring in oncogenes (BRAF, RAS) and those present at high frequency. Mutations more often missed in RNA‐Seq data involved tumor suppressor genes (TP53, PTEN) and when present at a lower frequency. None of the TERT mutations were identified by RNA‐Seq. This data suggest that RNA‐Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice.

RISKS OF PERFORMING HEMITHYROIDECTOMY WITH NECK DISSECTION IN MEDULLARY CANCER CASES. WHEN IT IS SAFE?

A. Semenov, D. Buzanakov, R. Chernicov, I. Sleptsov, V. Makarin, A. Uspenskaya, N. Timofeeva, I. Chinchuk, K. Novokshonov, J. Karelina, E. Fedorov, N. Gorskaya, I. Sablin, Y. Malugov, S. Elcheparova, A. Bubnov

Endocrine Surgery, SPbSU, Saint‐Petersburg, Russian Federation

Medullary thyroid cancer is recognized as rare and aggressive tumor derived from C‐cells. For about 30% to be an inherited forms. Standard initial treatment (ATA) is thyroidectomy with central compartment node dissection. But the risk of bilateral microscopic tumor in RET negative patients is minimal and safety benefits undoubtful. And also in most of cases patients deny to wait for RET test prior to operation. In 2009–2017 in North‐Western center of endocrinology and endocrine surgery (SPbSU) 229 patients underwent initial treatment for medullary thyroid cancer. In final histology multifocal tumor was reported in 46 cases (20.1%). Bilateral in 26 (11.4%). 1 of them had no RET mutation found. But in all cases multifocality was found and proofed by US and FNAB prior operation, except young patients undergoing “Prophylactic” thyroidectomy.

Only in 1 case with V804m RET mutation tumor was solitary. Two cases with multifocal unilateral tumor had C634F/S mutations. All of them were 15–18 y.o. at time of diagnosis and had relatives with MTC. But still OR for bilateral US involvement it prediction of familiar MTC was 437.

Tumor size and risk of multifocality. Tumor not bigger than 2 cm had 31 (67,4%) of patients with multifocal lesion. In group with solitary tumors 125 (67,8%) size was 2 cm and less. No difference found. In bilateral tumors 16 (61,5%) were 2 cm and less and 140 (69,5%) in unilateral. No difference found.

Local metastasis and risk of multifocality. No metastases were found in 22 (45,7%) multifocal cases and in 131 (71,5%) of unifocal cases. OR = 2,74. Probably due to intrathyroidal metastasis. 15 had no metastasis found (57,7%) in bilateral group and 138 (67,9%) in unilateral group respectively. OR = 1,45 probably due to earlier development of disease.

We also found that calcitonine to size (measured by US) ratio can predict multifocal lesions. ROC: AUC = 0,92. With cut‐off of 250 sensitivity was 85,7%, specificity – 87,5%. OR = 30. Hemithyroidectomy with neck dissection seems to be reasonable volume of initial surgery in any patient with no suspicious nodes in contralateral side, giving absolute diminishing of hypoparathyroidism risk.

IDENTIFICATION OF A FIVE‐GENE SIGNATURE AND ESTABLISHMENT OF A PROGNOSTIC NOMOGRAM TO PREDICT PROGRESSION‐FREE INTERVAL OF PAPILLARY THYROID CARCINOMA

M. Wu, Z. Liu

General Surgery, Peking Union Medical College Hospital, Beijing, China

Although prognosis of papillary thyroid carcinoma (PTC) is relatively good, it is important to select the minority of patients with poorer prognosis to avoid side effects in low‐risk patients associated with unnecessary over‐treatment; this requires accurate prognostic predictions. Six PTC expression datasets were obtained from the gene expression omnibus (GEO) database. Level 3 mRNA expression and clinicopathological data were obtained from The Cancer Genome Atlas Thyroid Cancer (TCGA THCA) database. Through integrated analysis of these datasets, highly reliable differentially‐expressed genes (DEGs) were identified and lasso Cox regression was applied to identify DEGs related to the progression‐free interval (PFI) and establish a prognostic gene signature. The performance of the five‐gene signature was evaluated. Tumor immunity was analyzed to investigate potential molecular mechanisms. Multivariate Cox regression analysis was used to identify factors associated with PTC prognosis. Finally, a prognostic nomogram was established. A novel five‐gene signature was established to predict the PTC PFI, including PLP2, LYVE1, FABP4, TGFBR3, and FXYD6, and the ROC curve and C‐index showed good performance in both training and validation datasets. This could classify patients into high‐ and low‐risk groups with distinct PFIs and differentiate PTC tumors from normal tissue. Univariate Cox regression revealed that this signature was an independent prognostic factor. The established nomogram, incorporating the prognostic gene signature and clinical parameters, was able to predict the PFI with high efficiency. The gene signature‐based nomogram was superior to the American Thyroid Association (ATA) risk stratification to predict PTC PFI. Tumor immunity analysis revealed differential tumor infiltration by immune cells between the high‐ and low‐risk groups. Moreover, lymphocytic infiltration was closely related to multiple immune‐related DEGs, which were possibly regulated by multiple transcription factors. Our study identified a five‐gene signature and established a prognostic nomogram, which were reliable in predicting the PFI of PTC; this could be beneficial for individualized treatment and medical decision making.

ADOPTION OF ACTIVE SURVEILLANCE: ANALYSIS OF A LARGE NATIONAL COHORT OF PHYSICIANS

N. Yang¹, M.C. Saucke¹, N. Marka¹, B. Hanlon¹, A.D. McDow², K.L. Long¹, S.C. Pitt¹

¹Department of Surgery, University of Wisconsin, Madison, WI; ²Department of Surgery, Indiana University School of Medicine, Indianapolis, IN

Adoption of active surveillance (AS) for papillary thyroid cancer (PTC) is in the early stages in the U.S. We aimed to characterize AS adoption nationally. We mailed a random sample of 1,500 providers in the American Medical Association a survey with 2 cases scenarios: 1) a 45yo female with a solitary, node‐negative 0.8cm PTC and no adverse features or history and 2) the same case but the patient preferred AS. We compared “non‐adopters” (who did not recommend AS) to “adopters” who recommended AS at least once using Fisher's exact and t‐tests. Of 488 responses (32.5%), 464 treated PTC and were analyzed (144 endocrinologists, 122 general surgeons, and 198 otolaryngologists). Respondents were 53.6 ± 9 yo and practiced 20.7 ± 9 yrs; 78.2% (n = 347) were male; 76.1% (n = 334) White. Non‐adopters (45.7%; n = 212) were significantly less likely than adopters to be in academics (17.2% vs 29.5%), see >25 PTC patients/yr (24.4% vs 42.2%), be aware AS is an option (79.8% vs 94.8%), or use ATA guidelines (80.0% vs 87.8%). Non‐adopters were also less likely to know if a patient is appropriate for AS (29.2% vs 49.8%), have resources to perform AS (46.2% vs 63.7%), want to increase AS use (6.5% vs 19.8%), believe AS is underused (66.7% vs 84.5%), or feel colleagues offer AS (1.1% vs 5.1%). Non‐adopters were more likely to have reservations about AS (37.5% vs 10.4%), agree AS makes them anxious (30.1% vs 7.6%), be concerned patients may have a poor outcome (34.9% vs 12.0%), and feel AS is a psychological burden for patients (48.1% vs 34.0%). We also compared “partial adopters” who only recommended AS if the patient preferred AS (42.2%; n = 196) to “full adopters” who recommended AS for both cases (11.4%; n = 53). Partial adopters were less likely to discuss AS in their practice (82.5% vs 94.3%) and more likely to have reservations about AS (12.9% vs 1.9%), be anxious about AS (9.3% vs 1.9%), and believe AS psychologically burdens patients (38.3% vs 18.9%). P < 0.05 for all comparisons. Efforts to increase AS adoption in the U.S. should focus on improving provider awareness, knowledge, and interest. Further evaluation of AS outcomes is needed to minimize provider uncertainty and should assess the psychological impact of AS on patients.

TEMPORAL REGULATION OF T₃‐DEPENDENT EPIGENETIC PROGRAMMING BY THYROID HORMONE RECEPTOR BETA IN THYROID CELLS

N.E. Gillis¹, J. Tomczak¹, S. Frietze², F. Carr¹

¹Pharmacology, University of Vermont, Burlington, VT; ²Biomedical and Health Sciences, University of Vermont, Burlington, VT

Nuclear hormone receptors control gene expression patterns in a highly tissue‐specific manner. Hormone‐dependent changes in local chromatin structure are a key regulatory component in this process. Thyroid hormone receptors (TRs) mediate the transcriptional effects of T₃ through the recruitment of co‐regulators and chromatin remodeling complexes. TRβ is the predominant TR isoform expressed in thyroid cells and has been shown to induce reorganization of chromatin and local nucleosome structures via interaction with genomic regulatory elements to alter target gene transcription. In this study, we have defined genome‐wide changes in chromatin compaction and gene expression in response to both short‐ (6 hours) and long‐term (24 hour) T₃ treatment using an integrated analysis of ATAC‐ and RNA‐seq data in the normal‐like human thyroid cell line Nthy‐ORI. Our RNA‐seq data indicates that T₃ elicits a dynamic transcriptional response in thyroid cells. 582 differentially expressed genes were detected after 6 hours of hormone treatment and 1,693 differentially expressed genes were detected after 24 hours. This is reflective of the changes in chromatin accessibility observed at these time points. The majority of T₃‐mediated gene expression changes are correlated with differentially accessible chromatin. In support of a role for TRβ in this regulation, thyroid hormone response elements were found to be enriched within ATAC‐seq peaks located in differentially accessible promoter regions. The differences between short‐ and long‐term changes in the chromatin landscape and associated gene expression in response to T₃ is likely due to accumulation of signals from initial hormone response, as evidenced by early changes in expression of upstream regulators and later changes in effectors of downstream pathways. Integration chromatin accessibility and gene expression reveals dynamic and temporally‐regulated T₃‐dependent transcriptional regulatory networks that are likely important for maintenance of normal thyroid epithelial cell identity. These findings can serve to enhance future studies aimed at understanding how hormone induced changes in chromatin structure occur in thyroid disease and thyroid cancer.

CRYO‐EM STRUCTURE OF THYROGLOBULIN

K. Kim¹, O. Clarke²

¹Pharmacology, Columbia University, New York, NY; ²Columbia University, New York, NY

Thyroglobulin (Tg) is a homodimeric iodoglycoprotein that is essential to thyroid hormonogenesis. After its production in thyrocytes, Tg is exported to the follicular lumen, where selected tyrosine residues within the protein molecule become iodinated to produce mono‐iodotyrosine (MIT) and di‐iodotyrosine (DIT) residues, which are substrates for oxidative coupling reactions that yield either T₄ (DIT and DIT) or T₃ (DIT and MIT). While the specificity of the iodination of tyrosine residues is thought to be due to the surface exposure of the tyrosine residue sites, the three‐dimensional structure of thyroglobulin remained elusive. Bovine thyroglobulin powder (Sigma‐Aldrich) were solubilized in buffer (10 mM HEPES pH7.4, 100 mM NaCl). The sample was loaded onto a TSKgel BioAssist G4SW_xl (Tosoh Bioscience) column. Purified Tg was eluted at 0.5 mL/min, and the combined sample was concentrated to 7 mg/mL. 0.2% CHAPS (GoldBio) was added to the sample.

3 μL of Tg sample was applied to the plasma‐cleaned holey‐gold grids. Grids were blotted for 7 s and vitrified by plunge freezing into liquid ethane with a Vitrobot (FEI). Data acquisition was done on a Titan Krios microscope (FEI) equipped with a K2 direct electron detector (Gatan) operated at 300 kV.

Micrographs were aligned using MotionCor2. Contrast transfer function parameters for each micrograph were obtained using CTFFIND. 554,053 particles were picked from 5016 micrographs to obtain the initial 3D map in cryoSPARC. Further refinement was performed in cryoSPARC and RELION3, which allowed de novo model builidng in Coot. We obtained a 3D reconstruction at 2.6 Å resolution and determined the structure of bovine Tg using single particle cryo‐EM, revealing the overall architecture including hormonogenic sites. Our results provide structural information of Tg type 1, 2, and 3 repeats as well as ChEL domain in Tg. Based on structural determination of specific donor and acceptor DIT and MIT residues, we found the distance between a donor Y130 and an acceptor Y5 would not allow physical interactions of the two hormonogenic iodotyrosine residues. Based on this structural observation, we hypothesize a coupling mechanism of two idodotyrosine residues to produce T₄.

GENOME‐WIDE ASSOCIATED STUDY OF BIOCHEMICAL RESIDUAL HYPOTHYROIDISM AFTER THYROIDECTOMY

L. Wang¹, S. Zhang², H. Luo¹, H. Xu², Z. Li¹

¹Department of Thyroid Surgery, West China Hospital of Sichuan University, Chengdu, Sichuan, China; ²Precision Medicine Center, State Key Laboratory of Biotherapy, Precision Medicine Key Laboratory of Sichuan Province, West China Hospital, Sichuan University and Collaborative Innovation Center, Sichuan, Chengdu, China

TSH suppression or replacement therapy is an important part of treatment for patients who received thyroidectomy surgery. But sometimes the patients would be beset by (subclinical) hypothyroidism, even clinical hypothyroidism by levothyroxine (L‐T4) monotherapy, which is defined as biochemical residual hypothyroidism (BRH). To investigate into the mechanism of BRH, we conducted genome‐wide associated study (GWAS) to identify the potential target loci.

Consecutive subjects were enrolled into the final study and divided into two cohorts (BRH and control cohorts). Subjects in BRH cohort defined as persistent high TSH level (>10 mU/L) over 3‐month prescription of L‐T4 or presented with obvert hypothyroidism symptoms. DNA was extracted from 5 ml peripheral blood of each subjects. Demographic, treatment history and thyroid function were recorded. GWAS was conducted in 162 subjects (BRH: n = 61 and control: n = 101). Mean age in BRH and control cohorts was 42.8 ± 13.5 and 40.3 ± 10.9 respectively. Mean value of thyroid‐stimulating hormone (TSH) between the two groups (BRH and control) was 19.9 ± 23.3 mU/L and 1.7 ± 4.0 mU/L (p < 0.001). GWAS result showed that the strongest associated SNPs was in HS3ST4 gene, rs75512357 (p = 8.2e‐6). And followed by rs727252 in PHF21B gene (2.9e‐5).rs75512357 in HS3ST4 gene may be associated with BRH. However, it needs to be validated in larger cohorts.

ASSOCIATION BETWEEN ATA SONOGRAPHIC RISK AND ACR TI‐RADS WITH THYROSEQ V2 NEXT‐GENERATION SEQUENCING IN THYROID NODULES WITH INDETERMINATE FINE NEEDLE ASPIRATION CYTOLOGY

S. Han¹, J. Ocampo¹, M. Arosemena¹, R. Kuker², P. Castillo², E. Scortegagna², A. Kargi³

¹Internal Medicine, Jackson Memorial Hospital, Miami, FL; ²Radiology, University of Miami, Miami, FL; ³Endocrinology, University of Miami, Miami, FL

Ultrasound risk stratification systems such as ACR TI‐RADS (TIRADS) and ATA sonographic risk (ATA‐US) can be used to risk stratify thyroid nodules with indeterminate FNA cytology and to determine which nodules should undergo molecular testing. For this purpose it would be helpful for clinicians to be informed about the association between sonographic risk categories and results of molecular tests. We performed a retrospective single center study including thyroid nodules with Bethesda III cytology that had undergone evaluation by Thyroseq v2 next‐generation sequencing between the years 2016–2017. Ultrasounds were classified by ATA‐US and TIRADS by 3 radiologists blinded to clinical data. ATA‐US categories included: a) high suspicion (HS), b) intermediate suspicion (IS), c) low suspicion (LS), d) very low suspicion (VLS) of malignancy or e) benign. TIRADS categories included: a) highly suspicious (TR5), b) moderately suspicious (TR4), c) mildly suspicious (TR3), d) not suspicious (TR2) or e) benign (TR1). Any mutation per Thyroseq was classified as a positive test.153 nodules met inclusion criteria of which 54 were Thyroseq positive.

Among Thyroseq positive nodules, 2 (4%) nodules were VLS, 14 (26%) were LS, 14 (26%) were IS and 24 (44%) were HS by ATA‐US. In the Thyroseq negative group, 1 (2%) was VLS, 36 (36%) were LS, 48 (48%) were IS and 14 (14%) were HS. The rate of Thyroseq positivity within each ATA‐US category was VLS 2%, LS 28%, IS 23% and HS 63%.

Regarding association of TIRADS and Thyroseq: Among Thyroseq positive nodules, 1 (2%) was TR1, 3 (6%) were TR2, 14 (26%) were TR3, 25 (46%) were TR4 and 11 (20%) were TR5. Within the Thyroseq negative group, 16 (16%) were TR2, 26 (26%) were TR3, 50 (51%) were TR4 and 7 (7%) were TR5. The rate of Thyroseq positivity within each TIRADS category was TR1 5%, TR2 16%, TR3 35%, TR4 33% and TR5 61%. These findings show an association between sonographic risk category and Thyroseq v2 results. Nodules with high suspicion ATA‐US and TIRADS are more likely to harbor mutations than nodules in the lower sonographic risk categories. We have also demonstrated that Thyroseq positivity is associated with higher sonographic risk by both ATA‐US and TIRADS.

MACROCALCIFICATIONS IN THYROID NODULES DO NOT ALTER MALIGNANCY RISK WITHIN ATA SONOGRAPHIC PATTERN SYSTEM (ATASPS)

K. Kobaly¹, C. Kim¹, J. Langer², S. Mandel¹

¹Endocrinology, University of Pennsylvania, Wynnewood, PA; ²Radiology, University of Pennsylvania, Philadelphia, PA

Microcalcifications and interrupted peripheral calcifications in hypoechoic nodules have a high suspicion (HS) of malignancy in the ATASPS. The impact of other calcification patterns on cancer risk is uncertain. We examined if non‐HS calcifications (NHSC, macrocalcifications and punctate echogenic foci in non‐hypoechoic nodules) modified cancer risk. A retrospective review of 728 consecutive biopsied thyroid nodules was performed. Each nodule was classified as Intermediate (IS), Low (LS) or Very low (VLS) suspicion pattern using ATASPS, or non‐classifiable. For nodules with NHSC, the nodule was assigned to an ATASPS based on its non‐calcified features. Cytology was analyzed for all nodules and surgical pathology results (n = 198) were assessed.101 of 728 (14%) nodules had NHSC. The ATASPS was IS (n = 18), LS (n = 62) and VLS (n = 21). The majority (n = 92, 91%) had large central or linear dystrophic calcifications; the remainder had punctate echogenic foci in non‐hypoechoic solid nodules (n = 9). The distribution of cytology diagnoses between NHSC and non‐calcified nodules within each ATASPS was the same (IS p = 0.13, LS p = 0.55, VLS p = 0.44). The estimated cancer prevalence of NHSC nodules was 7%, compared to 9% in the total group of IS/LS/VLS nodules. 9 of 62 (15%) LS nodules had punctate non‐shadowing echogenic foci, with an estimated cancer prevalence of 19% (vs 10% for total LS group, p = 0.24). In conclusion, non‐HS macrocalcifications did not alter malignancy risk predicted by ATASPS based upon other features.

PERFORMANCE OF ATA SONOGRAPHIC RISK AND ACR TI‐RADS ALONE AND IN COMBINATION WITH THYROSEQ V2 IN THE DIAGNOSIS OF THYROID NODULES WITH INDETERMINATE FNA CYTOLOGY

J. Ocampo¹, M. Arosemena¹, S. Han¹, R. Kuker², P. Castillo², E. Scortegagna², A. Kargi³

¹Department of Medicine, Jackson Memorial Hospital, Miami, FL; ²Department of Radiology, University of Miami, Miami, FL; ³Division of Endocrinology, Department of Medicine, University of Miami, Miami, FL

Molecular testing including Thyroseq genomic classifier is often utilized for diagnosis of thyroid nodules with indeterminate cytology (ITN). It has been proposed that ATA sonographic risk (ATA‐US) and ACR TI‐RADS (TIRADS) may be used to risk stratify ITNs. The aim of this study was to compare the diagnostic performance of ATA‐US and TIRADS with that of Thyroseq v2 (Tv2) and determine whether Thyroseq test performance is affected by sonographic risk. We performed a retrospective study of ITNs with Bethesda III cytology that underwent evaluation by Tv2 between years 2016–2017. Ultrasounds were reviewed by 3 radiologists and classified using ATA‐US and TIRADS. ATA‐US categories were benign, very low suspicion (VLS), low suspicion (LS), intermediate suspicion (IS) and high suspicion (HS) of malignancy. TIRADS included: benign (TR1), not suspicious (TR2), mildly suspicious (TR3), moderately suspicious (TR4) and highly suspicious (TR5). Nodules were deemed benign or malignant based on surgical pathology or a minimum 1‐year follow up by US or repeat FNA. Positive predictive values (PPV) and negative predictive values (NPV) were calculated. For ATA‐US, HS and IS were considered a positive test and LS and VLS a negative test; while for TIRADS TR3‐5 sonography were considered positive and TR1‐2 negative.86 nodules met inclusion criteria: 55 (64%) were deemed benign and 31 (36%), malignant. Rate of malignancy per ATA‐US category was 33% for VLS, 30% for LS, 31% for IS and 48% for HS. Per TIRADS, it was 0% for TR1, 14% for TR2, 41% for TR3, 39% for TR4 and 33% for TR5.

ATA‐US had a PPV of 38.3% and NPV of 69.2% while TIRADS had a PPV of 38.5% and NPV of 87.5%. Tv2 had an overall PPV of 61% and NPV of 90%. The PPV of Tv2 was similar across sonographic risk categories. NPV remained high at 87% in the higher sonographic risk nodules and was 100% in the lower sonographic risk ones. We conclude that in ITNs with Bethesda III cytology Tv2 test performance is superior to that of ATA‐US or TIRADS with the caveat that TIRADS may have a similar NPV to that of Tv2. Thyroseq test performance is not dependent on baseline sonographic risk, except for a slight increase in NPV when applied to nodules with low sonographic risk.

I‐TIRADS (INTERNATIONAL THYROID IMAGING, REPORTING, AND DATA SYSTEM) PROJECT: ROADMAP AND STATUS

F. Tessler

Radiology, University of Alabama at Birmingham, Birmingham, AL

At least eight guidelines for thyroid nodule risk‐stratification, mostly promulgated by professional organizations, are in active use. Although they all recommend which nodules warrant fine‐needle aspiration (FNA), a given nodule may be subjected to FNA, followed‐up, or ignored depending on the system used to classify it. The overall aim of the I‐TIRADS project is to develop a unified international system that adopts the best characteristics of existing guidelines. In May 2018, 16 physicians with special interest in thyroid nodule ultrasound and management took part in a teleconference and enthusiastically endorsed the aspirational goal of forming a group to develop universal guidelines. The International Thyroid Nodule Ultrasound Working Group (ITNUWG) is led by a steering committee of 10 experts representing seven societies. Subsequent efforts have focused on reaching consensus on a two‐phase project plan, recruiting additional physicians, and implementing methods for collaboration. Phase I is underway. Its primary goal is to create a lexicon of thyroid nodule ultrasound descriptors that discriminate between benignancy and malignancy; phase II will devise schemes to translate a nodule's descriptors to management guidance based on patterns, points, or a hybrid process, allowing physicians to choose whatever method suits them best.

Phase I is being conducted by four subgroups (composition/echogenicity, echogenic foci/macrocalcifications, shape/margins, and extra‐thyroidal extension/lymph nodes), each with five members. They are charged with performing literature searches, evaluating papers, and proposing descriptors in their categories. Consensus will be reached using a modified Delphi process. Phase II will begin by defining the malignancy risk ranges on which management guidelines will ultimately be based. Although overcoming organizational challenges has taken time, enthusiasm remains high, and we expect progress to accelerate over the coming months.