Non-Operative, Active Surveillance of Larger Malignant and Suspicious Thyroid Nodules.

CONTEXT: Active surveillance for papillary thyroid cancer (PTC) meeting criteria for surgical resection is uncommon. Which patients may prove reasonable candidates for this approach is not well defined. OBJECTIVE: To examine the feasibility and safety of active surveillance for patients with known or suspected intrathyroidal PTC up to 4cm in diameter. DESIGN, SETTING, AND PARTICIPANTS: Retrospective review of all consecutive patients who underwent non-operative active surveillance of suspicious or malignant thyroid nodules over a 20-year period from 2001-2021. We included patients with an initial US-FNA confirming either: a) Bethesda 5 or 6 cytology or, b) a "suspicious" AFIRMA molecular test. The primary outcomes and measures included the rate of adverse oncologic outcomes (mortality and recurrence), as well as the cumulative incidence of size/volume growth. RESULTS: Sixty-nine patients were followed with active surveillance for 1 year or longer (average 55 months), with 26 patients (38%) having nodules ≥ 2 cm. No patients were found to develop new incident occurrence of lymph node or distant metastasis. One patient however, demonstrated concern for progression to a dedifferentiated cancer on repeat core biopsy 17 years after initial start non-operative selection. 21% of patients had an increase in maximum diameter more than 3 mm, and volume increase ≥50% was noted in 25% of patients. Thirteen patients ultimately underwent delayed (rescue) surgery, and no disease recurrence was noted after such treatment. Age and initial nodule size were not predictors of nodule growth. CONCLUSIONS: These data expand consideration of active surveillance of papillary thyroid carcinoma in select patients with intrathyroidal suspected malignancy >1cm in diameter. Rescue surgery, if required at a later timepoint, appears effective.

Outcomes of cytologically indeterminate thyroid nodules managed with Genomic Sequencing Classifier.

CONTEXT: Molecular testing can refine the risk of malignancy in thyroid nodules with indeterminate cytology to decrease unnecessary diagnostic surgery. OBJECTIVE: This study was performed to evaluate the outcomes of cytologically indeterminate thyroid nodules managed with Afirma genomic sequencing classifier (GSC) testing. DESIGN, SETTING, PATIENTS, AND INTERVENTION: Adult patients who underwent a biopsy at three major academic centers between July 2017 and June 2021 with Bethesda III or IV cytology were included. All patients had surgery or minimum follow-up of 1 year ultrasound surveillance. MAIN OUTCOME MEASURES: The primary outcomes were the sensitivity, specificity, PPV, and NPV of GSC in Bethesda III and IV nodules. RESULTS: The median nodule size of the 834 indeterminate nodules was 2.1 cm and the median follow-up was 23 months. GSC's sensitivity, specificity, PPV, and NPV across all institutions were 95%, 81%, 50%, and 99% for Bethesda III nodules and 94%, 82%, 65%, and 98% for Bethesda IV nodules, respectively. The overall false negative rate was 2%. The NPV of GSC in thyroid nodules with oncocytic predominance was 100% in Bethesda III nodules and 98% in Bethesda IV nodules. However, the PPV of oncocytic nodules was low (17% in Bethesda III nodules and 45% in Bethesda IV nodules). Only 22% of thyroid nodules with benign GSC results grew during surveillance. CONCLUSIONS: GSC is a key tool for managing patients with indeterminate cytology, including the higher-risk Bethesda IV category. GSC benign thyroid nodules can be observed similarly to thyroid nodules with benign cytology.

Additional Oncogenic Alterations in RAS-Driven Differentiated Thyroid Cancers Associate with Worse Clinicopathologic Outcomes.

PURPOSE: RAS mutations occur across the spectrum of thyroid neoplasms, and more tools are needed for better prognostication. The objective of this study was to evaluate how additional genetic events affecting key genes modify prognosis in patients with RAS-mutant thyroid cancers, and specifically differentiated thyroid cancers (DTC). EXPERIMENTAL DESIGN: We performed a clinical-genomic analysis of consecutive patients with DTC, poorly differentiated (PDTC), or anaplastic thyroid cancer (ATC) between January 2014 and December 2021, in whom a custom-targeted next-generation sequencing assay was performed. Patients harboring RAS mutations were included, and we compared their clinical features and outcomes based upon the presence of additional oncogenic alterations. RESULTS: Seventy-eight patients were identified, with 22% (17/78) harboring a driver RAS mutation plus an additional oncogenic alteration. All six (100%) ATCs had an additional mutation. Compared with DTCs harboring a solitary RAS mutation, patients with DTC with RAS and additional mutation(s) were more likely to be classified as American Thyroid Association high-risk of recurrence (77% vs. 12%; P < 0.001) and to have larger primary tumors (4.7 vs. 2.5 cm; P = 0.002) and advanced stage (III or IV) at presentation (67% vs. 3%; P < 0.001). Importantly, over an average 65-month follow-up, DTC-specific-mortality was more than 10-fold higher (20% vs. 1.8%; P = 0.011) when additional mutations were identified. CONCLUSIONS: Identification of key additional mutations in patients with RAS-mutant thyroid cancers confers a more aggressive phenotype, increases mortality risk in DTC, and can explain the diversity of RAS-mutated thyroid neoplasia. These data support genomic profiling of DTCs to inform prognosis and clinical decision-making.

Papillary Thyroid Carcinomas with Tall Cell Features: An Intermediate Entity Between Classic and Tall Cell Subtypes.

Background: While the diagnosis of papillary thyroid carcinomas (PTCs) with tall cell features (PTCtcf) is often made for carcinomas with histological features intermediate between classic and tall cell subtypes of PTC (tcPTC), its comparative signature to that of either tcPTC or classic PTC is less clear. The objective of this study was to perform an integrative clinicopathologic and genomic analysis elucidating the spectrum of tcPTC, PTCtcf, and classic PTC. Methods: We analyzed all consecutive patients with tcPTC and PTCtcf evaluated at a tertiary academic referral center between 2005 and 2020, as well as a comparative cohort of classic PTC, in a retrospective observational cohort analysis. Clinicopathologic data were compared among the three groups, including progression-free survival (PFS), recurrent/persistent disease, and a negative composite outcome of death, progression, or need for advanced therapy. To specifically understand differences between tcPTC and PTCtcf, targeted next-generation sequencing was performed in a subset of these cohorts. Results: A total of 292 patients were analyzed (81 tcPTC, 65 PTCtcf, 146 classic PTC). Thirteen percent of tcPTC versus 8% of PTCtcf versus 1% of classic PTC had the advanced American Joint Committee on Cancer stage (p = 0.002). Similarly, macroscopic extrathyroidal extension was observed in 38% of tcPTC, 14% of PTCtcf, and 12% of classic PTC (p < 0.001). The 5-year PFS was 76.5%, 81.5%, and 88.3% for tcPTC, PTCtcf, and classic PTC, respectively, while the rates of the negative composite outcome 40.2% for tcPTC, 20.7% for PTCtcf, and 11.2% for classic PTC (p < 0.001). In a multivariable Cox regression analysis, the negative composite outcome was independently associated with tcPTC (HR 4.3 [confidence interval 1.1-16.1], p = 0.03). tcPTC demonstrated substantially more hotspot TERT promoter mutations than PTCtcf (44% vs. 6%, p = 0.012). Conclusions: Our study demonstrates a continuum of disease-specific risk of PTC, pointing at PTCtcf as an intermediate entity between tcPTC and classic PTC. These data provide a more refined understanding of risk at time of presentation, while better elucidating the diversity of genomic drivers.

Identifying and Predicting Diverse Patterns of Benign Nodule Growth.

CONTEXT: The natural history of benign thyroid nodules is typically characterized by slow growth and minimal risk of malignant transformation. Available data have, to date, been unable to elucidate the diversity of benign nodule growth patterns over time nor predictive of which patients follow which pattern. OBJECTIVE: We aimed to better define the diverse patterns of benign nodule behavior and their predictors. METHODS: We prospectively studied 389 consecutive patients with solitary, solid, cytologically benign thyroid nodules ≥1 cm and follow-up ultrasound for at least 4 years. Demographic, sonographic, biochemical data were collected at initial evaluation, and subsequent growth patterns were identified over the follow-up. Predictors of growth at initial evaluation and 3 years of follow-up were defined. RESULTS: The mean (±SD) follow-up was 7.7 (±2.7) years. Three distinct growth patterns were identified: A) stagnant nodules with average growth rate < 0.2 mm/year; B) slow-growing nodules with a rate 0.2 to 1.0 mm/year; and C) fast-growing nodules increasing > 1.0 mm/year. Fast-growing nodules represented 17.2% of the cohort, and were more frequent in patients younger than 50 years (OR 2.2 [1.2-4.1], P = 0.016), and in larger nodules (2.0-2.9 cm, OR 3.5 [1.7-7.1], P = 0.001; >3.0 cm, OR 4.4 [1.8-10.4], P = 0.001 vs reference 1-1.9 cm). In a multiple regression model, nodule growth at 3 years at an average growth rate over 0.2 mm/year over 3 years since initial evaluation was an independent predictor of longer-term fast nodule growth, even after adjusting for age, biological sex, TSH level, and nodule size (P < 0.001). CONCLUSION: The natural history of benign nodule growth is diverse, with over 80% of nodules demonstrating minimal to no growth long-term. Nearly 20% of cytologically benign nodules may exhibit a fast, continued growth pattern, which can be predicted by the 3-year growth rate pattern. These findings can help inform decision making for tailored benign nodule follow-up and monitoring.

Thyroid Nodule Shape Independently Predicts Risk of Malignancy.

CONTEXT: Predictive models of thyroid nodule cancer risk are presently based upon nodule composition, echogenicity, margins, and the presence of microcalcifications. Nodule shape has shown promise to be an additive factor helping determine the need for nodule biopsy. OBJECTIVE: We sought to determine if calculation of a nodule's spherical shape independently associates with cancer risk. METHODS: This prospective cohort study, conducted at a single large academic healthcare system in the United States, included patients with 1 or 2 clinically relevant thyroid nodules (predominantly solid and over 1 cm) presenting for diagnostic evaluation. Thyroid ultrasound, cytological evaluation with fine-needle biopsy, and/or histopathological examination on occasion of thyroid surgery were performed. We calculated the nodule's long to short ratio (spherical shape), and its association with tissue proven benign or malignant endpoints. RESULTS: The long to short nodule ratio was significantly lower in malignant compared to benign nodules indicating greater risk of malignancy in more spherical nodules (1.63 ±â€…0.38 for malignant nodules vs 1.74 ±â€…0.47 for benign, P < 0.0001). The risk of malignancy continually increased as the long to short ratio approached a purely spherical ratio of 1.0 (ratio > 2.00, 14.6% cancer; ratio 1.51-2.00, 19.7%; ratio 1.00-1.50, 25.5%, P < 0.0001). In multiple regression analysis, younger age, male sex, and nodule's spherical shape were each independently associated with cancer risk. CONCLUSION: The more a thyroid nodule is spherically shaped, as indicated by a long to short ratio approaching 1.0, the greater its risk of malignancy. This was independent of age, sex, and nodule size. Incorporating a nodule's sphericity in the risk stratification systems may improve individualized clinical decision making.

Immunotherapy-Associated Hypothyroidism: Comparison of the Pre-Existing With De-Novo Hypothyroidism.

Background: Immunotherapy has revolutionized the treatment of solid malignancies, but is associated with endocrine-related adverse events. This study aims to dissect the natural course of immunotherapy-induced hypothyroidism and provide guidance regarding diagnosis and management in patients with and without pre-existing hypothyroidism. Methods: A retrospective analysis was conducted using patients who received immunotherapy between 2010-2019 within a multicenter hospital system. Participants were separated in three groups-those with pre-existing hypothyroidism, those who developed primary hypothyroidism and those with hypophysitis within a year of their first immunotherapy. Serial effects of immunotherapy on thyroid function tests (TFTs) and levothyroxine dosing were evaluated. Results: 822 patients were screened, with 85 determined to have pre-existing hypothyroidism, 48 de-novo primary hypothyroidism and 12 de-novo hypophysitis. All groups displayed fluctuations in TFTs around weeks 6-8 of treatment. In the pre-existing hypothyroidism group, the levothyroxine dose was higher at 54 weeks than at baseline with the difference showing a trend towards statistical significance (p=0.06). The observed mean levothyroxine dose was significantly lower than the mean calculated weight-based dose for all groups. This finding was most clinically significant for the de-novo hypophysitis group (mean difference: -58.3 mcg, p<0.0001). The mean 0.9 mcg/kg levothyroxine dose at week 54 for the de-novo hypophysitis group was statistically lower than the other groups (p=0.009). Conclusion: It is reasonable to screen with TFTs every 4 weeks, and space out TFTs surveillance to every 12 weeks after week 20. Our findings suggest a more conservative approach for levothyroxine dosing in those developing de-novo hypothyroidism, especially hypophysitis, such as initiating at 0.9-1.2 mcg/kg.

Do Molecular Profiles of Primary Versus Metastatic Radioiodine Refractory Differentiated Thyroid Cancer Differ?

Management of metastatic radioiodine refractory differentiated thyroid cancer (DTC) can be a therapeutic challenge. Generally, little is known about the paired molecular profile of the primary tumor and the metastases and whether they harbor the same genetic abnormalities. The present study compared the molecular profile of paired tumor specimens (primary tumor/metastatic sites) from patients with radioiodine refractory DTC in order to gain insight into a possible basis for resistance to radioiodine. Twelve patients with radioiodine refractory metastases were studied; median age at diagnosis of 61 years (range, 25-82). Nine patients had papillary TC (PTC), one had follicular TC (FTC), and two had Hürthle cell TC (HTC). Distant metastases were present in the lungs (n = 10), bones (n = 4), and liver (n = 1). The molecular profiling of paired tumors was performed with a panel of 592 genes for Next Generation Sequencing, RNA-sequencing, and immunohistochemistry. Digital microfluidic PCR was used to investigate TERT promoter mutations. The genetic landscape of all paired sites comprised BRAF, NRAS, HRAS, TP53, ATM, MUTYH, POLE, and NTRK genes, including BRAF and NTRK fusions. BRAF V600E was the most common point mutation in the paired specimens (5/12). TERT promoter mutation C228T was detected in one case. PD-L1 expression at metastatic sites was highly positive (95%) for one patient with HTC. All specimens were stable for microsatellite instability testing, and the tumor mutation burden was low to intermediate. Therefore, the molecular profile of DTC primary and metastatic lesions can show heterogeneity, which may help explain some altered responses to therapeutic intervention.

Cytochrome C Oxidase Subunit 4 (COX4): A Potential Therapeutic Target for the Treatment of Medullary Thyroid Cancer.

The nuclear-encoded subunit 4 of cytochrome c oxidase (COX4) plays a role in regulation of oxidative phosphorylation and contributes to cancer progression. We sought to determine the role of COX4 in differentiated (DTC) and medullary (MTC) thyroid cancers. We examined the expression of COX4 in human thyroid tumors by immunostaining and used shRNA-mediated knockdown of COX4 to evaluate its functional contributions in thyroid cancer cell lines. In human thyroid tissue, the expression of COX4 was higher in cancers than in either normal thyroid (p = 0.0001) or adenomas (p = 0.001). The level of COX4 expression correlated with tumor size (p = 0.04) and lymph-node metastases (p = 0.024) in patients with MTCs. COX4 silencing had no effects on cell signaling activation and mitochondrial respiration in DTC cell lines (FTC133 and BCPAP). In MTC-derived TT cells, COX4 silencing inhibited p70S6K/pS6 and p-ERK signaling, and was associated with decreased oxygen consumption and ATP production. Treatment with potassium cyanide had minimal effects on FTC133 and BCPAP, but inhibited mitochondrial respiration and induced apoptosis in MTC-derived TT cells. Our data demonstrated that metastatic MTCs are characterized by increased expression of COX4, and MTC-derived TT cells are vulnerable to COX4 silencing. These data suggest that COX4 can be considered as a novel molecular target for the treatment of MTC.

Detection of BRAFV600E in Liquid Biopsy from Patients with Papillary Thyroid Cancer Is Associated with Tumor Aggressiveness and Response to Therapy.

The detection of rare mutational targets in plasma (liquid biopsy) has emerged as a promising tool for the assessment of patients with cancer. We determined the presence of cell-free DNA containing the BRAFV600E mutations (cfBRAFV600E) in plasma samples from 57 patients with papillary thyroid cancer (PTC) with somatic BRAFV600E mutation-positive primary tumors using microfluidic digital PCR, and co-amplification at lower denaturation temperature (COLD) PCR. Mutant cfBRAFV600E alleles were detected in 24/57 (42.1%) of the examined patients. The presence of cfBRAFV600E was significantly associated with tumor size (p = 0.03), multifocal patterns of growth (p = 0.03), the presence of extrathyroidal gross extension (p = 0.02) and the presence of pulmonary micrometastases (p = 0.04). In patients with low-, intermediate- and high-risk PTCs, cfBRAFV600E was detected in 4/19 (21.0%), 8/22 (36.3%) and 12/16 (75.0%) of cases, respectively. Patients with detectable cfBRAFV600E were characterized by a 4.68 times higher likelihood of non-excellent response to therapy, as compared to patients without detectable cfBRAFV600E (OR (odds ratios), 4.68; 95% CI (confidence intervals)) 1.26-17.32; p = 0.02). In summary, the combination of digital polymerase chain reaction (dPCR) with COLD-PCR enables the detection of BRAFV600E in the liquid biopsy from patients with PTCs and could prove useful for the identification of patients with PTC at an increased risk for a structurally or biochemically incomplete or indeterminate response to treatment.

Mitotane induces mitochondrial membrane depolarization and apoptosis in thyroid cancer cells.

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria­dependent metabolism has emerged as a promising strategy for thyroid cancer (TC) treatment. We hypothesized that mitotane targets mitochondria and induces apoptosis in TC cells. Cell lines representative of the major histological variants of TC were chosen: Follicular (FTC­133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0­100 µM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC­1 staining and by western blot analysis using an antibody against caspase­3. The expression of mitochondrial molecules and DNA damage markers and the activation of endoplasmic reticulum (ER) stress were determined by western blotting. The expression of mitochondrial ATP synthase subunit ß (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 µM for 24 h) decreased the viability of FTC­133, BCPAP, SW1736, C643 and TT cells by 12, 59, 54, 31 and 66%, respectively. Morphological evidence of ER stress and overexpression of ER markers was observed in TC cells following exposure to mitotane. The treatment led to increased expression of histone γH2AX, indicating DNA damage, and to caspase­3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro­apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine­protein kinase B­raf gene and proto­oncogene tyrosine­protein kinase receptor Ret. Treatment with mitotane was associated with loss of mitochondrial membrane potential and decreased expression of ATP5B, particularly in the medullary TC (MTC)­derived TT cells. Immunohistochemical analysis of mitochondrial ATP5B in human TC specimens demonstrated its overexpression in cancer compared with normal thyroid tissue. The level of ATP5B expression was higher in MTC compared with the follicular, papillary or anaplastic types of TC. Mitotane elicited pleiotropic effects on TC cells, including induction of ER stress, inhibition of mitochondrial membrane potential and induction of apoptosis. The results of the present study suggest that mitotane could be considered as a novel agent for the treatment of aggressive types of TC.

Association of Thyrotropin Suppression With Survival Outcomes in Patients With Intermediate- and High-Risk Differentiated Thyroid Cancer.

Importance: Suppression of thyrotropin (often referred to as thyroid-stimulating hormone, or TSH) with levothyroxine used in management of intermediate- and high-risk differentiated thyroid cancer (DTC) to reduce the likelihood of progression and death is based on conflicting evidence. Objective: To examine a cohort of patients with intermediate- and high-risk DTC to assess the association of thyrotropin suppression with progression-free survival (PFS) and overall survival. Design, Setting, and Participants: This cohort study used a multicenter database analysis including patients from tertiary referral centers and local clinics followed up for a mean (SD) of 7.2 (5.8) years. Patients with DTC treated uniformly with total thyroidectomy and radioactive iodine between January 1, 1979, and March 1, 2015, were included. Among the 1012 patients, 145 patients were excluded due to the lack of longitudinal thyrotropin measurements. Exposures: Levothyroxine therapy to target thyrotropin suppression with dose adjustments based on changing thyrotropin goal. Main Outcomes and Measures: The primary outcome measures were overall survival and PFS. A Cox proportional hazards model was used to assess the contribution of age, sex, tumor size, histology, and lymph node and distant metastases at landmarks 1.5, 3.0, and 5.0 years. The patients were divided into 3 groups based on mean thyrotropin score before each landmark: (1) suppressed thyrotropin, (2) moderately suppressed or low-normal thyrotropin, and (3) low-normal or elevated thyrotropin. Results: Among 867 patients (557 [64.2%] female; mean [SD] age, 48.5 [16.5] years) treated with a median (range) cumulative dose of 151 (30-1600) mCi radioactive iodine, disease progression was observed in 293 patients (33.8%), and 34 patients (3.9%) died; thus, the study was underpowered in death events. Thyrotropin suppression was not associated with improved PFS at landmarks 1.5 (P = .41), 3.0 (P = .51), and 5.0 (P = .64) years. At 1.5 and 3.0 years, older age (hazard ratio [HR], 1.06; 95% CI, 1.03-1.08 and HR, 1.05; 95% CI, 1.01-1.08, respectively), lateral neck lymph node metastases (HR, 4.64; 95% CI, 2.00-10.70 and HR, 4.02; 95% CI, 1.56-10.40, respectively), and distant metastases (HR, 7.54; 95% CI, 3.46-16.50 and HR, 7.10; 95% CI, 2.77-18.20, respectively) were independently associated with subsequent time to progression, while at 5.0 years, PFS was shorter for patients with lateral neck lymph node metastases (HR, 3.70; 95% CI, 1.16-11.90) and poorly differentiated histology (HR, 71.80; 95% CI, 9.80-526.00). Conclusions and Relevance: Patients with intermediate- and high-risk DTC might not benefit from thyrotropin suppression. This study provides the justification for a randomized trial.

Detection at Public Facilities of 131I in Patients Treated for Differentiated Thyroid Cancer: Frequency, Sites, Management by Security Agents, and Physician Documentation Recommended for Patients.

Patients treated with 131I may be identified at security checkpoints at various public facilities. The objective of this survey was to determine the frequency of detection, the spectrum of public facilities, the various methods of management of the situation by security agents, and the spectrum of physician documentation for patients regarding their 131I therapy. Methods: Data were tabulated from a Thyroid Cancer Survivors' Association, Inc., survey emailed to approximately 15,000 associates and available online from December 2013 to December 2014. Responses were tabulated from respondents who reported that they were 18 y old or older, had received at least 1 131I treatment for differentiated thyroid cancer, and were responding regarding their last 131I treatment. Results: Of 621 respondents, 595 reported an attempt to pass through a public facility security checkpoint. Of these 595 patients, approximately 10% (57) were identified as being radioactive. The facility reported by 43 respondents was an airport for 35% (15), border crossing for 33% (14), government building for 19% (8), shopping mall for 7% (3), train station for 5% (2), and steel recycling plant for 2% (1). The security agent's management of the situation reported by 47 respondents included questioning for 81% (38), allowing them to proceed without a change in travel plans for 57% (27), requesting documentation of the therapy for 55% (26), rescanning for 55% (26), calling a member of the treating team for validation for 17% (8), "strip" searching for 4% (2), detaining such that a change in travel plans was required for 6% (3), and prohibiting continued travel for 4% (2). The period of detainment reported by these 47 respondents was less than 30 min for 57% (27), 30 to less than 60 min for 21% (10), 1 to less than 1.5 h for 15% (7), 1.5 to less than 2 h for 2% (1), 2-4 h for 0% (0), and greater than 4 h for 4% (2). Data regarding physician documentation are presented. Conclusion: The detection of radioactivity at a variety of security checkpoints at public facilities after131I therapy occurred in approximately 10% of respondents. Travel inconvenience is not infrequent and may require alteration of travel plans. Physicians should take steps to ensure that patients not only have appropriate documentation of their 131I therapy with them but also have instructions regarding how security agents may verify their 131I therapy.

Nelfinavir inhibits proliferation and induces DNA damage in thyroid cancer cells.

The HIV protease inhibitor Nelfinavir (NFV) inhibits PI3K/AKT and MAPK/ERK signaling pathways, emerging targets in thyroid cancers. We examined the effects of NFV on cancer cells that derived from follicular (FTC), papillary (PTC) and anaplastic (ATC) thyroid cancers. NFV (1-20 µM) was tested in FTC133, BCPAP and SW1736 cell lines. The effects of NFV on cell proliferation were determined in vitro using real-time microscopy and by flow cytometry. DNA damage, apoptotic cell death and expression of molecular markers of epithelial-mesenchymal transition (EMT) were determined by Western blot and real-time PCR. Real-time imaging demonstrated that NFV (10 µM) increased the time required for the cell passage through the phases of cell cycle and induced DNA fragmentation. Growth inhibitory effects of NFV were associated with the accumulation of cells in G0/G1 phase, downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). NFV also induced the expression of γH2AX and p53BP1 indicating DNA damage. Treatment with NFV (20 µM) resulted in caspase-3 cleavage in all examined cells. NFV (20 µM) decreased the levels of total and p-AKT in PTEN-deficient FTC133 cells. NFV had no significant effects on total ERK and p-ERK in BRAF-positive BCPAP and SW1736 cells. NFV had no effects on the expression of EMT markers (Twist, Vimentin, E- and N-Cadherin), but inhibited the migration and decreased the abilities of thyroid cancer cells to survive in non-adherent conditions. We conclude that NFV inhibits proliferation and induces DNA damage in thyroid cancer cell lines. Our in vitro data suggest that NFV has a potential to become a new thyroid cancer therapeutic agent.

18F-FDG-PET SUV AS A PROGNOSTIC MARKER OF INCREASING SIZE IN THYROID CANCER TUMORS.

OBJECTIVE: Positron emission tomography/computed tomography (PET/CT) scans with 2-[fluorine-18] fluoro-2-deoxy-D-glucose (18F-FDG) are used in high-risk thyroid cancer patients to identify metastasis. The prognostic significance of increases in standardized uptake values (SUVs) has not been clearly defined. This pilot study investigated the correlation between SUV increases and subsequent changes in individual lesion size. METHODS: A retrospective chart review of patients with histologically confirmed thyroid cancer who were monitored with serial 18F-FDG-PET/CT scans from 2008 to 2013 was performed. Forty-seven patients were selected for analysis. A mixed-effects statistical model was used after data normalization. RESULTS: For a 10% increase in SUV, a 6% increase in tumor area was observed (P<.0001). Analysis on cube root-transformed data from serial scans was significant in 4 of 5 groups: scans 1 to 2 (P = .0001), scans 2 to 3 (P = .0005), scans 3 to 4 (P = .008), scans 4 to 5 (P = .66), and overall (P<.0001). After exclusion of outliers, for a 10% increase in SUV, the expected percentage increases in area on subsequent scans were found to be 3.4% (P = .0006), 2.6% (P = .005), 4% (P = .074), and 4.1% (P = .27) for the second, third, fourth, and fifth scans, respectively. The association was similarly significant in cases with a ≥25% increase in SUV. Secondary analysis showed a significant association of SUV with thyroglobulin (Tg) level (P = .035) but not with thyroid-stimulating hormone (TSH) level (P = .85). CONCLUSIONS: A significant positive correlation was noted between the increase in lesional SUV and subsequent increase in lesion area. An increase in lesional SUV in subsequent scans may portend tumor growth and could prompt consideration for earlier or more aggressive intervention. ABBREVIATIONS: DTC = differentiated thyroid cancer EORTC = European Organization for Research and Treatment of Cancer 18F-FDG = 2-[fluorine-18] fluoro-2-deoxy-D-glucose FNA = fine-needle aspiration MTC = medullary thyroid cancer PET/CT = positron emission tomography/computed tomography PVE = partial volume effect RAI = radioactive iodine SUV = standardized uptake value Tg = thyroglobulin TSH = thyroid-stimulating hormone.

Targeted therapies in thyroid cancer: an extensive review of the literature.

INTRODUCTION: Patients with progressive, metastatic, RAI-refractory differentiated thyroid cancer (DTC), as well as patients with advanced medullary (MTC) and anaplastic thyroid cancer represent a cohort for which therapeutic options are limited. The recent discoveries in the molecular mechanisms implicated in TC have provided insight of the pathogenesis and progression of disease. In that respect, targeted therapies have emerged as a promising alternative for the treatment of those patients. Areas covered: Tyrosine Kinase Inhibitors (TKIs) have been studied extensively in TC: sorafenib and lenvatinib have been approved by the FDA for the treatment of metastatic, RAI-refractory DTC, while vandetanib and cabozantinib are FDA approved for use in advanced MTC. Moreover, several additional TKIs, multi-targeted or specific, are currently under investigation in TC. The current manuscript provides an extensive review of the literature regarding targeted therapies in TC including the rationale behind their use, the clinical trials and an expert opinion on their use. Literature in English appearing at PubMed was thoroughly reviewed, especially manuscripts of the last 5 years. Expert commentary: Patients with advanced, progressive, metastatic TC should be evaluated for enrollment in a clinical trial or should be placed on treatment with one of the FDA- and EMA- approved agents.

Effects of Dosimetrically Guided I-131 Therapy on Hematopoiesis in Patients With Differentiated Thyroid Cancer.

OBJECTIVE: The objective of the study was to evaluate the effects of dosimetrically guided I-131 prescribed activities on hematopoiesis reflected by changes in complete blood counts (CBCs). DESIGN: This was a retrospective analysis. SETTING: The study was conducted at an academic center. PATIENTS: A total of 152 patients with differentiated thyroid cancer who had 185 dosimetrically guided I-131 treatments. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Repeated-measure ANOVA was used for the analysis of the differences in the averages of CBCs that were documented at baseline and 1, 6, 12, 24­36, and 48­60 months after I-131 treatment. RESULTS: All parameters decreased to their respective nadir at 1 month and then gradually returned toward baseline values. White blood cells (WBCs) and platelets (PLTs) were the most significantly affected cells. At 1 month, the decrease was 29.6% (P < .0001) for WBCs and 25% (P < .0001) for PLTs, whereas at 12 months, the decrease was 15.5% (P < .0001) and 13% (P < .0001), respectively. Lymphocytes appeared to be more susceptible to I-131 than neutrophils (ANCs). The decreases were small in absolute numbers for red blood cells, hematocrit and hemoglobin not surpassing 10%. Multivariate analysis demonstrated that the ratio of administered prescribed activity-to-maximum tolerated activity was associated with the decreases in WBCs (P = .0038), ANCs (P = .0063), and red blood cells (P = .029), with borderline significance for PLTs (P = .057) and hemoglobin (P = .057). CONCLUSIONS: Dosimetrically guided I-131 resulted in statistically significant decreases in CBC parameters, which were more prominent in WBCs and PLTs. Lymphocytes were more severely affected than ANCs, whereas all parameters reached a nadir at 1 month and then gradually returned toward baseline values over the 5-year follow-up of our study.

Phase 2 clinical trial of sunitinib as adjunctive treatment in patients with advanced differentiated thyroid cancer.

OBJECTIVE: Our objective was to evaluate the efficacy and safety of sunitinib following at least one course of radioactive iodine treatment in patients with advanced differentiated thyroid cancer (DTC). The study endpoints included best response rate (including best objective response rate) and progression-free survival (PFS) per Response Evaluation Criteria in Solid Tumors (RECIST) 1.1, measurement of serum thyroglobulin (Tg), and toxicity evaluation. DESIGN AND METHODS: This was a single center, nonrandomized, open-label, phase 2 clinical trial. In total, 23 patients were enrolled and were treated with a starting daily, oral dose of 37.5  mg sunitinib. Patients were evaluated with imaging, laboratory tests, and physical examination periodically per protocol. RESULTS: The mean best response was a decrease of 17.2% (S.D. 22.8) in tumor sum from baseline. Six (26%) patients achieved a partial response (PR), and 13 (57%) had stable disease (SD) for a clinical benefit rate (PR+SD) of 83%. The overall median PFS was 241 days (interquartile limits, 114-518). No statistically significant difference was observed between the medians of the baseline and post-treatment Tg values (P=0.24). The most common adverse events included grades 1 and 2 decreases in blood cell counts (especially leukocytes), diarrhea, fatigue, hand-foot skin reaction, nausea, musculoskeletal pain, and hypertension. CONCLUSIONS: These data demonstrate that sunitinib exhibits significant anti-tumor activity in patients with advanced DTC. Since sunitinib was relatively well-tolerated, there is the potential for clinical benefit in these patients, and further investigation of this agent is warranted.

Metformin Attenuates 131I-Induced Decrease in Peripheral Blood Cells in Patients with Differentiated Thyroid Cancer.

BACKGROUND: 131I treatment (tx) of differentiated thyroid cancer (DTC) is associated with hematopoietic toxicity. It was hypothesized that metformin could have radioprotective effects on bone-marrow function. The objective was to determine whether metformin prevents 131I-induced changes in complete blood counts (CBC) in patients with DTC. METHODS: A retrospective analysis was performed of CBC values in DTC patients who were (40 patients: metformin group) or were not taking metformin (39 patients: control group) at the time of administration of 131I. Repeated measures analysis of variance was used for the analysis of the differences in the averages of CBC that were documented at baseline and at 1, 6, and 12 months post 131I tx. RESULTS: The groups were comparable in terms of age, sex, stage of DTC, 131I dose administered, and baseline CBC values. In the control group, the decrease in white blood cells (WBC) was 35.8% (p < 0.0001) at one month, 21.8% (p < 0.0001) at six months, and 19.4% (p < 0.0001) at 12 months. In the metformin group, the decrease in WBC was 17.1% (p < 0.0001) at one month, and 8.6% at six months (p = 0.01), while at 12 months WBC had returned to baseline values (p = 0.9). Differences between the two groups were highly statistically significant at all time points (p < 0.0001, p = 0.0027, and p < 0.0001, respectively). Lymphocytes were more sensitive to 131I, but metformin's radioprotective properties were more prominent in neutrophils. At 12 months, the decrease in platelets in the control group was 15.5% (p < 0.0001) versus 5.6% (p = 0.056) in the metformin group, while at one and six months the reductions in the two groups were comparable. No statistically significant differences were observed between the two groups in the change from baseline values for hemoglobin. CONCLUSIONS: Metformin attenuated the 131I-induced decrease in CBC parameters, and its radioprotective properties were more prominent in WBC. Patients who were taking metformin during 131I tx also experienced a faster recovery in their blood counts, when compared to the control group. Further study is warranted in order to examine if the radioprotective properties of metformin observed in the current study for 131I tx can also apply to other forms of therapeutic chemo- and radiotherapy.

Glucose-deprivation increases thyroid cancer cells sensitivity to metformin.

Metformin inhibits thyroid cancer cell growth. We sought to determine if variable glucose concentrations in medium alter the anti-cancer efficacy of metformin. Thyroid cancer cells (FTC133 and BCPAP) were cultured in high-glucose (20 mM) and low-glucose (5 mM) medium before treatment with metformin. Cell viability and apoptosis assays were performed. Expression of glycolytic genes was examined by real-time PCR, western blot, and immunostaining. Metformin inhibited cellular proliferation in high-glucose medium and induced cell death in low-glucose medium. In low-, but not in high-glucose medium, metformin induced endoplasmic reticulum stress, autophagy, and oncosis. At micromolar concentrations, metformin induced phosphorylation of AMP-activated protein kinase and blocked p-pS6 in low-glucose medium. Metformin increased the rate of glucose consumption from the medium and prompted medium acidification. Medium supplementation with glucose reversed metformin-inducible morphological changes. Treatment with an inhibitor of glycolysis (2-deoxy-d-glucose (2-DG)) increased thyroid cancer cell sensitivity to metformin. The combination of 2-DG with metformin led to cell death. Thyroid cancer cell lines were characterized by over-expression of glycolytic genes, and metformin decreased the protein level of pyruvate kinase muscle 2 (PKM2). PKM2 expression was detected in recurrent thyroid cancer tissue samples. In conclusion, we have demonstrated that the glucose concentration in the cellular milieu is a factor modulating metformin's anti-cancer activity. These data suggest that the combination of metformin with inhibitors of glycolysis could represent a new strategy for the treatment of thyroid cancer.