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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.

Abstract 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.

Abstract 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. Methods Adult patients who underwent a biopsy at 3 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. The primary outcomes were the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (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 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. Conclusion 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.

Abstract 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 This work aimed to examine the feasibility and safety of active surveillance for patients with known or suspected intrathyroidal PTC up to 4 cm in diameter. Methods A retrospective review was conducted of all consecutive patients who underwent nonoperative active surveillance of suspicious or malignant thyroid nodules over a 20-year period from 2001 to 2021. We included patients with an initial ultrasound–fine-needle aspiration 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 or larger. 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 of nonoperative selection. A total of 21% of patients had an increase in maximum diameter more than 3 mm, while volume increase of 50% or greater 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. Conclusion These data expand consideration of active surveillance of PTC in select patients with intrathyroidal suspected malignancy greater than 1 cm in diameter. Rescue surgery, if required at a later time point, appears effective.

Mitotane is used for the treatment of adrenocortical cancer and elicits its anticancer effects via inhibition of mitochondrial respiration. Targeting mitochondria-dependent 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 (FTC-133), poorly differentiated (BCPAP), anaplastic (SW1736 and C643) and medullary (TT) TC cells, and were treated with mitotane (0-100 μM). Mitochondrial membrane potential, cell viability and apoptosis were examined by JC-1 staining and by western blot analysis using an antibody against caspase-3. 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 β (ATP5B) was examined by immunostaining in 100 human TC tissue samples. Treatment with mitotane (50 μM for 24 h) decreased the viability of FTC-133, 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 caspase-3 cleavage. Consistent with the results of the cell viability assays, the overexpression of pro-apoptotic genes following treatment with mitotane was more prominent in TC cells harboring mutations in the serine/threonine-protein kinase B-raf gene and proto-oncogene tyrosine-protein 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.

Abstract Disclosure: A. Bikas: None. E. Su: None. H. Patankar: None. M. Alshalalfa: Veracyte, Inc. Y. Hao: Veracyte, Inc. J.P. Klopper: Veracyte, Inc.. T. Pappa: None. Introduction: Alterations in cell cycle regulatory genes may lead to abnormal cellular proliferation, tumor development, and malignancy. The aim of this study was to characterize the expression of cell cycle proliferation genes in thyroid nodules/cancer and assess the molecular and clinical associations of their expression. Methods: A set of 47 genes implicated in cell cycle progression (CCP) genes (i.e. TOP2A, MKI67) were identified from The Cancer Genome Atlas (TCGA) Thyroid. A CCP activity z-score was derived from the expression of the 47 genes and subsequently associated with genomic alterations and outcomes data in TCGA. In addition, 2,205 fine needle aspiration (FNA) samples with (B)ethesda V/VI cytology sent for Afirma testing were extracted from the Afirma thyroid nodule database. The CCP score was analyzed in reference to TERT promoter mutation status and common oncogenic alterations reported by the Afirma Xpression Atlas (XA - the variant and fusion panel), as well as other molecular markers of tumor aggressiveness. Fisher’s exact test was used to assess statistical differences. Results: TCGA samples were stratified based on the CCP score and then grouped into 4 quartiles (Q4: top 25%, Q3:50-75%, Q2:25-50%, Q1: low 25%). Comparing Q4 to Q1, Q4 was enriched with TERT promoter mutations (14.4% vs 2.4%, p<0.001), disease progression (20% vs 8%, p=0.001), MACIS>8 (9.6% vs 3.2%, p=0.06), and stage IV disease (13.6% vs5.6%, p=0.05). The Q4 group is associated with a shorter time to disease progression (HR:2.56, 95%CI [1.23-5.3], p=0.01) relative to the Q1 group. In Afirma B V/VI samples, Q4 CCP score was more enriched with TERT promoter mutations (11.2% vs 4.9%, p p<0.001) compared to Q1, but less enriched with BRAFV600E (40% vs 69%, p<0.001), and RAS family variants (1.8% vs 6.4%, p<0.001). Within this subset, CCP was negatively correlated with TDS (r= -0.48, p<0.001) but not correlated with ERK activity or BRAF-RAS score. Mutations in genes of the PI3K/AKT/mTOR pathway were present, though in small numbers, in Q3 and Q4 groups, but not in Q1-Q2 groups. The CCP score was not correlated with sex or age. Conclusion: The CCP score may be a useful tool in pre-operative thyroid tumor samples to predict tumor aggressiveness, especially in the absence of known molecular alterations or in intermediate risk mutations (such as BRAFV600E) that present with a heterogeneous histologic phenotype. Interestingly, the highest CCP quartile, associated with worse progression free survival in the TCGA cohort, was less enriched with BRAFV600E compared to the lowest CCP quartile. Future analysis incorporating pathology and recurrence outcomes will be necessary to assess the validity and clinical utility of the preoperative CCP score. Presentation: Monday, July 14, 2025

Disclosure: G.A. Stamatiades: None. A. Bikas: None. H.J. Shah: None. K. Wong: None. A. Vaidya: None. M. Nehs: None. S.S. Basaria: None. Background: Pheochromocytomas are rare catecholamine-secreting tumors that arise from sympathetic adrenomedullary chromaffin tissue. Rarely, pheochromocytomas co-secrete ACTH and patients present with Cushing syndrome. Due to its rarity, clinicians may not consider this etiology of Cushing syndrome. We report a case of a man with ACTH-producing pheochromocytoma in whom methodic systematic approach led to its diagnosis and successful treatment. Clinical Case: A 48-year-old man presented with altered mental status. His family confirmed depressed mood, paucity of thought, and occasional blank stares. The patient was diagnosed with type-2 diabetes mellitus and hypertension 3 months ago, which were not controlled despite being on multiple anti-hypertensives and anti-diabetic agents. His EEG, head CT, and brain MRI were normal. On admission, his laboratory tests were notable for hypokalemia (3.0 mmol/L, n<5.3 mmol/L) and metabolic alkalosis (HCO3 33 mmol/L, n<32 mmol/L). Physical examination showed facial plethora, multiple ecchymoses and profound proximal muscle weakness. CT scan of the abdomen showed a 3.4 cm left adrenal nodule with a density of 33 Hounsfield units and a 73% absolute washout. The right adrenal gland was hyperplastic. Plasma and urinary metanephrines were significantly elevated (plasma metanephrines 1.6 nmol/l, n < 0.50 nmol/l, urinary metanephrines 1,685 mcg/24h, n <646 mcg/24h). Midnight salivary cortisol was 11,500 ng/dl (n<100 ng/dl) and 24h urinary free cortisol was 13,099 mcg/24h (n < 45 mcg/24h). ACTH was 450 pg/ml (n<63pg/ml) with a serum cortisol of 93 ug/dl (n<18.4 ug/dl). His serum cortisol after 8 mg Dexamethasone Suppression test was 100 ug/dl. DOTATATE PET CT scan showed uptake in the left adrenal nodule. The workup was suggestive of left-sided pheochromocytoma producing ectopic ACTH. The patient underwent laparoscopic left adrenalectomy. Postoperative ACTH was undetectable, indicating surgical cure. His blood pressure and plasma glucose gradually improved and remained normal after discontinuation of all medications. Surgical pathology was consistent with pheochromocytoma with positive staining for ACTH, confirming the diagnosis of ACTH-producing pheochromocytoma. He was discharged home after successful rehabilitation. Clinical Lessons: Pheochromocytomas are a rare cause of ectopic ACTH production, resulting in clinical Cushing syndrome. Imaging of the contralateral adrenal gland provides a valuable clue in making the diagnosis of ectopic ACTH production (as the gland is enlarged rather than atrophic). Reference: (1) Elliott PF, Berhane T, Ragnarsson O, Falhammar H. Ectopic ACTH- and/or CRH-Producing Pheochromocytomas. J Clin Endocrinol Metab. 2021 Jan 23;106(2):598-608. doi: 10.1210/clinem/dgaa488. PMID: 32710791. Presentation: Friday, June 16, 2023

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.

T. Pappa: None. H. Patankar: None. A.N. Bikas: None. K. Sehgal: None. A.S. Alqahtani: None. J. Park: None. B. Reardon: None. S. Rodig: None. K. Pfaff: None. S. Jones: None. N. Besson: None. R. Haddad: None. E. Van Allen: None. E.K. Alexander: None. Background: Thyroid cancer (TC) is the most common endocrine malignancy, and in the last decade our understanding of its genomic landscape has significantly advanced. Yet, information about the immune microenvironment of TC and its association with prognosis is limited. This is important particularly in advanced disease, where systemic therapies, including immunotherapy, may be of benefit. Hypothesis: Given the interaction between tumor compartment and its microenvironment, we hypothesized that oncogenic drivers of thyroid cancer may be associated with distinct immune microenvironmental states.Methods: We assessed patients with TC, who received care at Brigham and Women’s Hospital and Dana-Farber Cancer Institute (DFCI), and in whom the DFCI OncoPanel (panel sequencing of 447 cancer genes) and ImmunoProfile (CD8, FOXP3, PD1, PDL1 staining) assays were performed on their tumor sample. We performed an immune-genomic correlation seeking to understand the link between oncogenic drivers of TC and immune biomarker expression. Chi-square test and one-way ANOVA were used for statistical analyses between categorical variables.Results: We studied 21 patients with both OncoPanel and ImmunoProfile annotations available. Mean age was 59.7 years, 52% were females. The cohort consisted of 7 patients with papillary TC, 1 follicular TC, 2 oncocytic TC, 8 anaplastic TC, 1 medullary TC and 1 NUT carcinoma. The prevalence of BRAFV600E mutation was 42.8% and of RAS (N/K/H) 23.8%. Four of 21 tumors had a TERT promoter mutation, 8 a variant in PI3K/AKT/mTOR pathway and 2 a variant within the SWI/SNF complex. Patients with RAS mutant tumors were more likely to have low CD8 expression (p=0.007), FOXP3 (p=0.009), PD1 (p=0.007) and PDL1 eIC (area of PDL1+inflammatory cells) (0.007). There was no significant association between immune activity and patient’s sex, BRAFV600 mutational status or TERT promoter mutation. In a larger cohort of 60 patients including non-thyroid solid tumors and available genomic and ImmunoProfile data, we similarly observed that RAS mutant tumors were more likely to have low CD8, FOXP3 and PD1 expression (p <0.05 for all markers), suggesting that the RAS-related immune pattern possibly extends beyond thyroid cancer. Conclusions: To our knowledge this is the first study illustrating that RAS mutant thyroid tumors have a distinct immune devoid profile. This might guide interpretation of our recent clinical trial (NCT03246958) findings that NRAS mutation was associated with worse outcomes after immunotherapy with ipilimumab/nivolumab (1). Validation of this immunogenomic relationship in larger thyroid cohorts will inform the role of the immune microenvironment in molecular subtypes of thyroid cancer. 1.Genomic correlates of response to dual immune checkpoint inhibition in advanced thyroid carcinoma. World Congress on Thyroid Cancer Academy 6/17/2023 London UK; 388169:139. Monday, June 3, 2024

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. 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. 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. Levothyroxine therapy to target thyrotropin suppression with dose adjustments based on changing thyrotropin goal. 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. 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). Patients with intermediate- and high-risk DTC might not benefit from thyrotropin suppression. This study provides the justification for a randomized trial.