Risk factors for recurrent disease in small papillary thyroid cancers - a Swedish register-based study.

AIMS: To study the correlation between clinicopathological risk factors and the risk for intervention-requiring cancer recurrence in patients with small papillary thyroid cancers (sPTCs). MATERIALS AND METHODS: Records for 397 patients with sPTC (T1 ≤ 20mm) were obtained from the Scandinavian Quality Register for Thyroid, Parathyroid and Adrenal Surgery (SQRTPA) between 2010 and 2016. Follow-up time was at least 5 years. Data regarding intervention-requiring cancer recurrence were obtained from patient medical records and analysed regarding lymph node (LN) status (N0, N1a and N1b) and recurrence. RESULTS: Age was significantly lower in the N1a and N1b groups compared to N0 (45 vs. 40.5 vs. 49 years, respectively; p = 0.002). Tumour size was smaller in the N1a group compared to N1b group (9 vs. 11.8 mm; p <0.01). The mean number of metastatic LNs at initial surgery was higher in the N1b compared to N1a group (6.6 vs. 3; p = 0.001), and in the recurrent compared to the non-recurrent group (7 versus 3.9; p <0.01). The recurrence rate was higher in the N1b group than the N1a and N0 groups (25% vs. 2.4% vs. 1.4%, respectively; p = 0.001). CONCLUSIONS: Lymph node stage N1b at diagnosis, and having five or more metastatic nodes, are strong risk factors for cancer recurrence and decreased disease-free survival in sPTC. The management of patients with sPTC should include thorough lymph node mapping for optimal treatment and individual risk stratification.

Change in Practice of Radioactive Iodine Administration in Differentiated Thyroid Cancer: A Single-Centre Experience.

OBJECTIVE: Our study aimed to analyse temporal trends in radioactive iodine (RAI) treatment for thyroid cancer over the past decade; to analyse key factors associated with clinical decisions in RAI dosing; and to confirm lower activities of RAI for low-risk patients were not associated with an increased risk of recurrence. METHODS: Retrospective analysis of 1,323 patients who received RAI at a quaternary centre in Australia between 2008 and 2018 was performed. Prospectively collected data included age, gender, histology, and American Joint Committee on Cancer stage (7th ed). American Thyroid Association risk was calculated retrospectively. RESULTS: The median activities of RAI administered to low-risk patients decreased from 3.85 GBq (104 mCi) in 2008-2016 to 2.0 GBq (54 mCi) in 2017-2018. The principal driver of this change was an increased use of 1 GBq (27 mCi) from 1.3% of prescriptions in 2008-2011 to 18.5% in 2017-2018. In patients assigned as low risk per ATA stratification, lower activities of 1 GBq or 2 GBq (27 mCi or 54 mCi) were not associated with an increased risk of recurrence. In patients assigned to intermediate- or high-risk categories who received RAI as adjuvant therapy, there was no difference in risk of recurrence between 4 GBq (108 mCi) and 6 GBq (162 mCi). CONCLUSIONS: Our data demonstrate an evolution of RAI activities consistent with translation of ATA guidelines into clinical practice. Use of lower RAI activities was not associated with an increase in recurrence in low-risk thyroid cancer patients. Our data also suggest lower RAI activities may be as efficacious for adjuvant therapy in intermediate- and high-risk patients.

Prophylactic central lymph node dissection informs the decision of radioactive iodine ablation in papillary thyroid cancer.

BACKGROUND: Prophylactic central lymph node dissection (CLND) in papillary thyroid cancer (PTC) is controversial. We aimed to investigate if prophylactic CLND aids risk stratification and contributes to the decision for postoperative RAI ablation. METHODS: Patients undergoing thyroidectomy for PTC and prophylactic CLND were identified from an endocrine surgical unit database. Pathology reports where reviewed for number and size of lymph nodes and patients stratified by risk according to the ATA guidelines. RESULTS: 426 patients were identified with PTC ≤4 cm and prophylactic CLND. 96 patients (23%) had central lymph node metastasis (CLNM) that qualified them for the intermediate risk group. In 17 patients (4%), the CLNM data led to upgrading independently of other histopathological characteristics. Correcting for multiple variables, CLNM was an independent factor contributing to RAI treatment. CONCLUSION: Prophylactic CLND provides information to aid the selection of RAI ablation independent of primary cancer histology for risk stratification in 4% of patients. This benefit should be carefully balanced with the risk of CLND and patient treatment choice when deciding on management of PTC ≤4 cm.

Molecular Markers Guiding Thyroid Cancer Management.

The incidence of thyroid cancer is rapidly increasing, mostly due to the overdiagnosis and overtreatment of differentiated thyroid cancer (TC). The increasing use of potent preclinical models, high throughput molecular technologies, and gene expression microarrays have provided a deeper understanding of molecular characteristics in cancer. Hence, molecular markers have become a potent tool also in TC management to distinguish benign from malignant lesions, predict aggressive biology, prognosis, recurrence, as well as for identification of novel therapeutic targets. In differentiated TC, molecular markers are mainly used as an adjunct to guide management of indeterminate nodules on fine needle aspiration biopsies. In contrast, in advanced thyroid cancer, molecular markers enable targeted treatments of affected signalling pathways. Identification of the driver mutation of targetable kinases in advanced TC can select treatment with mutation targeted tyrosine kinase inhibitors (TKI) to slow growth and reverse adverse effects of the mutations, when traditional treatments fail. This review will outline the molecular landscape and discuss the impact of molecular markers on diagnosis, surveillance and treatment of differentiated, poorly differentiated and anaplastic follicular TC.

Biochemistry may be misleading in metachronous MEN2A-associated phaeochromocytoma following unilateral total adrenalectomy.

A 63-year-old woman with multiple endocrine neoplasia type 2A (MEN2A) presented with recurrent spells of headaches, sweats and palpitations decades after right adrenalectomy for phaeochromocytoma, and total thyroidectomy for medullary thyroid cancer. She was hypertensive and in sinus rhythm. DOTA-TATE positron-emission tomography (PET) demonstrated a 12mm enhancing left adrenal incidentaloma. 24 hours urine catecholamines, and multiple plasma metanephrine and normetanephrine measurements were all within normal reference ranges. Based on her symptoms and imaging findings, left adrenalectomy was performed and found a 40 mm phaeochromocytoma. Her symptoms have since completely resolved and plasma metanephrine is now undetectable MEN2-associated phaeochromocytomas are often bilateral and may be metachronous. Patients at high risk of phaeochromocytoma who develop symptoms of catecholamine excess should be carefully evaluated even if plasma or urinary metanephrines are within the normal reference range. Biochemical reference ranges for metanephrines need to be adjusted accordingly in patients who have had prior unilateral total adrenalectomy.

Short-term low-calorie diet remodels skeletal muscle lipid profile and metabolic gene expression in obese adults.

Diet intervention in obese adults is the first strategy to induce weight loss and improve insulin sensitivity. We hypothesized that improvements in insulin sensitivity after weight loss from a short-term dietary intervention tracks with alterations in expression of metabolic genes and abundance of specific lipid species. Eight obese, insulin-resistant, nondiabetic adults were recruited to participate in a 3-wk low-calorie diet intervention cohort study (1,000 kcal/day). Fasting blood samples and vastus lateralis skeletal muscle biopsies were obtained before and after the dietary intervention. Clinical chemistry and measures of insulin sensitivity were determined. Unbiased microarray gene expression and targeted lipidomic analysis of skeletal muscle was performed. Body weight was reduced, insulin sensitivity [measured by homeostatic model assessment of insulin resistance, (HOMA-IR)] was enhanced, and serum insulin concentration and blood lipid (triglyceride, cholesterol, LDL, and HDL) levels were improved after dietary intervention. Gene set enrichment analysis of skeletal muscle revealed that biosynthesis of unsaturated fatty acid was among the most enriched pathways identified after dietary intervention. mRNA expression of PDK4 and MLYCD increased, while SCD1 decreased in skeletal muscle after dietary intervention. Dietary intervention altered the intramuscular lipid profile of skeletal muscle, with changes in content of phosphatidylcholine and triglyceride species among the pronounced. Short-term diet intervention and weight loss in obese adults alters metabolic gene expression and reduces specific phosphatidylcholine and triglyceride species in skeletal muscle, concomitant with improvements in clinical outcomes and enhanced insulin sensitivity.

IL6 and LIF mRNA expression in skeletal muscle is regulated by AMPK and the transcription factors NFYC, ZBTB14, and SP1.

Adenosine monophosphate-activated protein kinase (AMPK) controls glucose and lipid metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells, and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce Il6 or leukemia inhibitory factor ( Lif) mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors that may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors nuclear transcription factor Y subunit c ( Nfyc), specificity protein 1 ( Sp1), and zinc finger and BTB domain containing 14 ( Zbtb14), or AMPK α1/α2 subunits, increased constitutive levels of Il6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK, Nfyc, Sp1, and Zbtb14 as novel regulators of immunometabolic signals from skeletal muscle.

FAK tyrosine phosphorylation is regulated by AMPK and controls metabolism in human skeletal muscle.

AIMS/HYPOTHESIS: Insulin-mediated signals and AMP-activated protein kinase (AMPK)-mediated signals are activated in response to physiological conditions that represent energy abundance and shortage, respectively. Focal adhesion kinase (FAK) is implicated in insulin signalling and cancer progression in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism. METHODS: Human muscle was treated with insulin or the AMPK-activating compound 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing the FAK gene, PTK2. RESULTS: AMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397 in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated by increased p-ACCS222, concomitant with reduced p-FAKY397. FAK signalling was reduced owing to serum starvation and AICAR treatment as demonstrated by reduced p-paxillinY118. Silencing PTK2 in primary human skeletal muscle cells increased palmitate oxidation and reduced glycogen synthesis. CONCLUSIONS/INTERPRETATION: AMPK regulates FAK signalling in skeletal muscle. Moreover, siRNA-mediated FAK knockdown enhances lipid oxidation while impairing glycogen synthesis in skeletal muscle. Further exploration of the interaction between AMPK and FAK may lead to novel therapeutic strategies for diabetes and other chronic conditions associated with an altered metabolic homeostasis.

Insulin and Glucose Alter Death-Associated Protein Kinase 3 (DAPK3) DNA Methylation in Human Skeletal Muscle.

DNA methylation is altered by environmental factors. We hypothesized that DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation of selected genes was determined in muscle from healthy men and men with type 2 diabetes before and after a glucose tolerance test. Insulin altered DNA methylation in the 3' untranslated region of the calcium pump ATP2A3 gene. Insulin increased DNA methylation in the gene body of DAPK3, a gene involved in cell proliferation, apoptosis, and autophagy. DAPK3 methylation was reduced in patients with type 2 diabetes. Carbohydrate ingestion reduced DAPK3 DNA methylation in healthy men and men with type 2 diabetes, suggesting glucose may play a role. Supporting this, DAPK3 DNA methylation was inversely correlated with the 2-h glucose concentration. Whereas glucose incorporation to glycogen was unaltered by small interfering RNA against DAPK3, palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating that DNA methylation constitutes a rapidly adaptive epigenetic mark. Furthermore, insulin and glucose modulate DAPK3 DNA methylation in a reciprocal manner, suggesting a feedback loop in the control of the epigenome.

Altered promoter methylation of PDK4, IL1 B, IL6, and TNF after Roux-en Y gastric bypass.

BACKGROUND: Early benefits of Roux-en Y gastric bypass (RYGB) are partly mediated by the caloric restriction that patients undergo before and acutely after the procedure. Altered DNA methylation occurs in metabolic diseases including obesity, as well as in skeletal, muscle eight months after RYGB. The objective of this study was to test whether promoter methylation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1 A), pyruvate dehydrogenase kinase isozyme-4 (PDK4), transcription factor A (TFAM), interleukin-1 beta (IL1 B), interleukin-6 (IL6) and tumor necrosis factor-α (TNF) is altered in blood after a very low calorie diet (VLCD) or RYGB. METHODS: Obese nondiabetic patients (n = 18, body mass index [BMI] 42.3 ± 4.9 kg/m(2)) underwent a 14-day VLCD followed by RYGB. Nonobese patients (n = 6, BMI 25.7 ± 2.1 kg/m(2)) undergoing elective cholecystectomy served as controls. DNA methylation of selected promoter regions was measured in whole blood before and after VLCD. A subgroup of seven patients was studied 1-2 days and 12 ± 3 months after RYGB. Promoter methylation was measured using methylated DNA capture and quantitative real-time polymerase chain reaction (PCR). RESULTS: VLCD decreased promoter methylation of PPARGC1 A. Methylation of PPARGC1 A, TFAM, IL1 B, IL6, and TNF promoters was changed two days after RYGB. Similar changes were also seen on day one after cholecystectomy. Moreover, methylation increased in PDK4, IL1 B, IL6, and TNF promoters 12 months after RYGB. CONCLUSION: RYGB induced more profound epigenetic changes than VLCD in promoters of the tested genes in whole blood. Changes in DNA methylation may contribute to the improved overall metabolic health after RYGB.

Downregulation of diacylglycerol kinase delta contributes to hyperglycemia-induced insulin resistance.

Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.

MEF2 activation in differentiated primary human skeletal muscle cultures requires coordinated involvement of parallel pathways.

The myocyte enhancer factor (MEF)2 transcription factor is important for development of differentiated skeletal muscle. We investigated the regulation of MEF2 DNA binding in differentiated primary human skeletal muscle cells and isolated rat skeletal muscle after exposure to various stimuli. MEF2 DNA binding activity in nonstimulated (basal) muscle cultures was almost undetectable. Exposure of cells for 20 min to 120 nM insulin, 0.1 and 1.0 mM hydrogen peroxide, osmotic stress (400 mM mannitol), or 1.0 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) led to a profound increase in MEF2 DNA binding. To study signaling pathways mediating MEF2 activity, we preincubated human skeletal muscle cell cultures or isolated rat epitrochlearis muscles with inhibitors of p38 mitogen-activated protein kinase (MAPK) (10 microM SB-203580), MEK1 (50 microM PD-98059), PKC (1 and 10 microM GF109203X), phosphatidylinositol (PI) 3-kinase (10 microM LY-294002), or AMP-activated protein kinase (AMPK; 20 microM compound C). All stimuli resulted primarily in activation of MEF2D DNA binding. Exposure of cells to osmotic or oxidative stress increased MEF2 DNA binding via pathways that were completely blocked by MAPK inhibitors and partially blocked by inhibitors of PKC, PI 3-kinase, and AMPK. In epitrochlearis muscle, MAPK inhibitors blocked contraction but not AICAR-mediated MEF2 DNA binding. Thus activation of MEF2 in skeletal muscle is regulated via parallel intracellular signaling pathways in response to insulin, cellular stress, or activation of AMPK.