United States

Background: The Choosing Wisely campaign from the American Board of Internal Medicine (ABIM) Foundation has scrutinized the practice of routinely ordering tests that offer limited or no benefit to patients, and have the potential to cause harm. Although postoperative day 1 (POD1) hormonal testing is necessary for certain patients who undergo adrenalectomy (i.e. those with functional tumors), the clinical utility of routinely checking POD1 non-endocrine labs is unclear, particularly in patients with non-functional adenomas. We sought to investigate how often these non-endocrine labs were abnormal after adrenalectomy and whether they represent value-added care.
Methods: Patients who underwent open or laparoscopic adrenalectomy at our institution from 2011-2020 were identified, and those with non-endocrine serum labs (hemoglobin, potassium, creatinine, or glucose) obtained before 0800 on POD1 were analysed. The frequency of abnormal labs, and any associated intervention(s), was recorded. Exclusion criteria varied by lab type and included: preoperative hemoglobin &lt;8 g/dL or intraoperative blood transfusion (hemoglobin analysis), presence of chronic kidney disease (potassium and creatinine analyses), diagnosis of primary aldosteronism (potassium analysis), and pheochromocytoma or diabetes mellitus (glucose analysis). Costs were calculated using the 2020 Medicare Clinical Laboratory Fee Schedule.
Results: Of 936 patients (56% female) who underwent adrenalectomy (92% laparoscopic), 91% (n=847) had ≥1 POD1 non-endocrine lab drawn. Only 0.4% (n=3) of 785 eligible patients had an abnormal POD1 hemoglobin (&lt;8 g/dL), and 1 patient received a blood transfusion. Potassium abnormalities were present in 3.4% (n=21) of 624 eligible patients, including 9 with hypokalemia (&lt;3.5 mmol/L) and 12 with mild hyperkalemia (5.5-5.9 mmol/L). Specific interventions for hypo/hyperkalemia were required in 5 patients (0.8% of all potassium values). AKI (creatinine increase of 0.3 g/dL or ≥1.5x baseline) was diagnosed in 4% of 759 eligible patients (n=28) and all were stage 1. Hyperglycemia (&gt;180 mg/dL) was identified in 1% (n=6) of 577 eligible patients, and 1 patient received short-acting insulin. Of a total of 2,745 lab values, while only 2% were abnormal, the total aggregate cost of routine POD1 labs was ~$17,000 during the study period.
Conclusions: Routine POD1 hemoglobin and creatinine testing are rarely abnormal after adrenalectomy and infrequently impact clinical management. Although patients with functional disease may require monitoring of electrolytes and/or glucose on POD1, in the current era of value-based healthcare, routine ordering of these labs may be unnecessary in selected patients with non-functioning tumors.

ENDO 2022

Challenging the Status Quo: Are Routine Post-Operative Day 1 Non-Endocrine Labs Necessary After Adrenalectomy?

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Background: The Choosing Wisely campaign from the American Board of Internal Medicine (ABIM) Foundation has scrutinized the practice of routinely ordering tests that offer limited or no benefit to patients, and have the potential to cause harm. Although postoperative day 1 (POD1) hormonal testing is necessary for certain patients who undergo adrenalectomy (i.e. those with functional tumors), the clinical utility of routinely checking POD1 non-endocrine labs is unclear, particularly in patients with non-functional adenomas. We sought to investigate how often these non-endocrine labs were abnormal after adrenalectomy and whether they represent value-added care.
Methods: Patients who underwent open or laparoscopic adrenalectomy at our institution from 2011-2020 were identified, and those with non-endocrine serum labs (hemoglobin, potassium, creatinine, or glucose) obtained before 0800 on POD1 were analysed. The frequency of abnormal labs, and any associated intervention(s), was recorded. Exclusion criteria varied by lab type and included: preoperative hemoglobin <8 g/dL or intraoperative blood transfusion (hemoglobin analysis), presence of chronic kidney disease (potassium and creatinine analyses), diagnosis of primary aldosteronism (potassium analysis), and pheochromocytoma or diabetes mellitus (glucose analysis). Costs were calculated using the 2020 Medicare Clinical Laboratory Fee Schedule.
Results: Of 936 patients (56% female) who underwent adrenalectomy (92% laparoscopic), 91% (n=847) had ≥1 POD1 non-endocrine lab drawn. Only 0.4% (n=3) of 785 eligible patients had an abnormal POD1 hemoglobin (<8 g/dL), and 1 patient received a blood transfusion. Potassium abnormalities were present in 3.4% (n=21) of 624 eligible patients, including 9 with hypokalemia (<3.5 mmol/L) and 12 with mild hyperkalemia (5.5-5.9 mmol/L). Specific interventions for hypo/hyperkalemia were required in 5 patients (0.8% of all potassium values). AKI (creatinine increase of 0.3 g/dL or ≥1.5x baseline) was diagnosed in 4% of 759 eligible patients (n=28) and all were stage 1. Hyperglycemia (>180 mg/dL) was identified in 1% (n=6) of 577 eligible patients, and 1 patient received short-acting insulin. Of a total of 2,745 lab values, while only 2% were abnormal, the total aggregate cost of routine POD1 labs was ~$17,000 during the study period.
Conclusions: Routine POD1 hemoglobin and creatinine testing are rarely abnormal after adrenalectomy and infrequently impact clinical management. Although patients with functional disease may require monitoring of electrolytes and/or glucose on POD1, in the current era of value-based healthcare, routine ordering of these labs may be unnecessary in selected patients with non-functioning tumors.

technical paper

Background: Pheochromocytomas and paragangliomas (PPGLs) have the highest degree of heritability among endocrine tumors. Currently, ~40% of PPGL individuals have a genetic germline pathogenic variant and exist at least 12 different genetic syndromes related to these tumors. Pathogenic variants in the Succinate Dehydrogenase Complex Subunit B (SDHB) gene account for about 10% of PPGL cases. Moreover, SDHB pathogenic variants are the most well-established risk factor to predict metastatic disease (40%-50% of cases). Germline SDHB large deletions are very rare worldwide, but SDHB exon 1 deletion has been reported in patients with PPGLs from Portugal and Spain. Indeed, a putative founder effect for SDHB exon 1 deletion was suggested in PPGL patients from Iberian Peninsula. Aim: To investigate a putative founder effect for SDHB exon 1 deletion. Methods: Eighteen unrelated Brazilian patients with germline heterozygous SDHB pathogenic variants were included. Additionally, two unrelated individuals with SDHB exon 1 complete deletion from Argentina were studied. SDHB pathogenic variants were investigated by automated SAGER sequencing, multiplex ligation-dependent probe amplification (SALSA MLPA Probemix P226 SDH) and/or high-throughput sequencing. Five SDHB flanking microsatellite markers at chromosome 1p (D1S2697, GATA29A05, D1S2826, D1S2644, and D1S199) were used to investigate if patients carrying this deletion have a common origin. Haplotypes were reconstructed using the PHASE algorithm (v. 2.1). A control group comprising 26 unrelated Brazilian individuals was also studied. Results: Among 18 Brazilian patients with germline SDHB pathogenic variants, heterozygous SDHB exon 1 complete deletion was identified in 6 of them (33% of the cases). The remaining 12 patients presented intragenic SDHB pathogenic variants without hotspot location. All Brazilian index patients with SDHB exon 1 deletion presented with paraganglioma, located mostly in the abdomen (4 abdominal; one thoracic; two head and neck and one colonic). Median age was 31.5 years and metastatic disease occurred in 3 (50%) of them. Haplotype analysis showed that 4 apparently unrelated Brazilian patients (4 out of 6 cases, 67%) shared a common allele (SDHB-GATA29A05-D1S2826-D1S2644-D1S199 | SDHB-186-130-213-102), which was not seen in chromosomes without the SDHB exon 1 deletion (p= 0.01). The two cases from Argentina did not have this haplotype, suggesting that SDHB exon 1 deletion in Argentina have a different origin. Conclusion: SDHB exon 1 complete deletion was the most frequent SDHB defect in our cohort. Our findings indicate a founder effect for SDHB exon 1 complete deletion in Brazilian patients with paraganglioma.
Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6

Evidence for a Founder Effect of SDHB Exon 1 Complete Deletion in Brazilian Patients with Paraganglioma

Introduction: Frailty is characterized by multisystem function decline related to aging and can be measured using frailty Index (FI). FI is constructed based on the proportion of deficits present in an individual out of the total number of health variables considered. FI in various populations has been shown to predict adverse health outcomes. We aimed to determine the impact of hormone excess on frailty in a prospective cohort of patients with adrenal adenomas.
Methods: We conducted a multicenter prospective observational study of adult patients with adrenal adenomas and referent subjects without adrenal disorders (Jan 2019-Dec 2021). Adrenal adenomas comprised non-functioning adrenal adenomas (NFA), adenomas with mild autonomous cortisol secretion (MACS), defined as dexamethasone suppression test (DST)>1.8 mcg/dL, Cushing syndrome (CS), and primary aldosteronism (PA). FI was calculated as a sum of 47 variables (20 comorbidities, 13 symptoms, 14 activities of daily living) divided by 47.¹ Frailty was defined as FI>0.25. In a subset of patients (n=176), functional measurements (handgrip strength, timed up and go test, gait speed) were performed and compared to FI.
Results: Of 233 patients (72.1% women), 71 were diagnosed with NFA (median age 64.5 years), 108 with MACS (median age 60.7 years), 11 with CS (median age 58.0 years), 46 with PA (median age 52.4 years), and 33 were referent subjects (median age 51.3 years). When compared to referent subjects, age- and sex-adjusted frailty prevalence was highest in CS (OR 29, 95%CI 4.3-202), followed by MACS (OR 16, 95%CI 3.5-73.7), and PA (OR 11.7, 95%CI 2.4-56), but not in patients with NFA (OR 5.1, 95%CI 0.99-25.8). Compared to patients with NFA, age- and sex-adjusted frailty prevalence was higher in CS (OR 5.8, 95%CI 1.4-24.4) and in MACS (OR 3.2, 95%CI 1.5-6.7), but not in PA (OR 2.3, 95%CI 0.9-6.1) group. Subgroup-analysis based on DST demonstrated a similar increase in age- and sex-adjusted frailty prevalence in patients with DST>5 mcg/dL (OR 3.1, 95%CI 1.2-8.2) and DST 1.9-5 mcg/dL (OR 3.3, 95%CI 1.5-7.4) when compared to patients with DST<1.8 mcg/dL. Higher FI was associated with lower handgrip measurement (P<0.0001), longer time during timed up and go test (P <0.0001), and decreased gait speed (P<0.0001).
Conclusions: Patients with functional adrenal adenomas are more likely to be frail. In patients with MACS, the prevalence of frailty was not associated with the degree of DST abnormality. FI correlated with currently validated but more cumbersome functional measurements and is a valuable tool that could be easily used in clinical practice and help individualize management decisions in patients with adrenal adenomas.
¹ Singh S et al. Frailty in Patients With Mild Autonomous Cortisol Secretion is Higher Than in Patients with Nonfunctioning Adrenal Tumors. JCEM 2020

Increased Frailty in Patients With Functioning and Nonfunctioning Adrenal Adenomas

ARMC5 is a tumor suppressor gene responsible for 20 to 40% of Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) with a function that remains unclear. Based on pathway analysis from RNAseq results obtained on zebrafish models of transient Armc5 up- and down-regulation, we identified transcriptional alterations of several members of SIRT1 (sirtuin (silent mating type information regulation 2 homolog) 1) signaling in our models and hypothesized that ARMC5 can regulate SIRT1 and its signaling in adrenocortical cells. Accordingly, the expression of the desacetylase SIRT1 is significantly increased in PBMAH tissues mutated for ARMC5 compared to tumors without mutations. However, this overexpression of SIRT1 is associated with an elevation of the profile of acetylated protein in the absence of ARMC5 suggesting that SIRT1 activity is actually decreased and that SIRT1 expression could increase to maintain its activity. Consistently, the measurement of SIRT activity on PBMAH tissues demonstrate a decrease of its activity when ARMC5 is mutated. Similar results are obtained in adrenal cells of 18-month-old Armc5+/- mice that are also hyper-corticosteronemic. Altogether, these data support that ARMC5 could regulate SIRT1 expression and/or activity. In vitro measurement of purified SIRT1 activity in the presence of ARMC5-enriched protein extracts demonstrated that the presence of ARMC5 protein does indeed alter SIRT1 activity. We hypothesize, therefore, that ARMC5 may be a new regulator of SIRT1 function but the underlying mechanism and the consequences of abnormal acetylated proteins on adrenocortical function require further investigation.

ARMC5 as a possible regulator of acetylation in the adrenal cortex in partnership with SIRT1

Introduction
In critically ill adults, high plasma free cortisol in face of suppressed plasma ACTH coincides with high plasma pro-opiomelanocortin (POMC), the ACTH precursor (1). Further augmenting the systemic glucocorticoid availability with hydrocortisone treatment further lowered plasma ACTH, while plasma POMC remained unaffected (2). The dynamics of POMC in relation to ACTH and free cortisol prior to intensive care unit (ICU) admission are unknown, as well as the impact hereon of glucocorticoid treatment. Also, elevated plasma POMC concentrations have not yet been confirmed in pediatric ICU (PICU) patients. In pediatric cardiac surgery-induced critical illness, we hypothesized that plasma POMC is elevated and that plasma ACTH transiently rises per-operatively and becomes suppressed once free cortisol has risen on PICU day 1 and 2. In addition, we hypothesized that in patients receiving glucocorticoids per-operatively, plasma ACTH is further suppressed while plasma POMC is unaffected.
Methods
From 53 children aged 0-36 months, who underwent cardiac surgery and were admitted to the PICU, blood samples were taken prior to surgery (pre-operatively, available samples N=51), during surgery (per-operatively, N=47), on the following morning (PICU day 1, N=40) and the day hereafter (PICU day 2, N=24). Blood samples were also collected from 24 age- and gender-matched healthy children. To investigate the impact of synthetic glucocorticoid treatment on the endogenous plasma hormone concentrations, patients were categorized into steroid-naive (total N=38) and steroid-treated patients (total N=15). Plasma hormone concentrations were quantified with highly-specific ELISA (POMC), RIA (ACTH, cortisol, CBG) or colorimetric assays (albumin). Cross reactivity with synthetic glucocorticoids within the used cortisol RIA is minimal (<0.27% for methylprednisolone, <0.1% for dexamethasone). Free cortisol was estimated with the adapted Coolens' formula.
Results
Plasma POMC was increased pre-operatively (p<0.0001) but no longer thereafter (p>0.05), irrespective of steroid treatment. Plasma ACTH in patients was never higher than in healthy children. While in steroid-naive patients, plasma ACTH became suppressed only by PICU day 1 (p<0.0001), steroid-treated patients had already suppressed plasma ACTH per-operatively (p≤0.0001). In steroid-naive patients, plasma free cortisol was increased from per-operatively onwards (p≤0.002), while in steroid-treated patients, plasma free cortisol (endogenous) concentrations always remained comparable to those of healthy children (p>0.05).
Conclusion
Pre-operatively high plasma POMC, not followed by increased ACTH, suggests a centrally-activated HPA-axis already prior to surgery and an immature pituitary processing of POMC into ACTH. Systemic glucocorticoid availability is elevated from surgery onwards, likely driven by ACTH-independent mechanisms. Further increasing systemic glucocorticoid availability exogenously with glucocorticoid administration accelerates the suppression of plasma ACTH. The long-term impact of early glucocorticoid treatment in critically ill children remains to be investigated.
References
(1) Téblick A. et al. Critical Care 2021.
(2) Téblick A. et al. Endocrinology 2022.

Pro-opiomelanocortin (POMC), ACTH and Cortisol Responses to Pediatric Cardiac Surgery

Purpose. To evaluate deep learning analysis of thyroid nodule ultrasound images as a rule-out test for thyroid malignancy.
Methods. Supervised deep learning (DL) classifier of thyroid nodules was trained on 32,545 thyroid US images from 621 nodules representing all major benign and malignant types of thyroid lesions and tested on an independent set of 145 nodules collected at a different healthcare system in the United States. The Big Transfer BiT-M ResNet-50x1 convolutional neural net architecture was modified to contain 3, 4, 6 and 3 PreActBottleneck units per block 1 through 4. Weights pretrained on the ImageNet-21k dataset were loaded and weights for blocks 3 and 4 were fine-tuned for the binary classification task of distinguishing benign and malignant thyroid nodules.
Results. The deep learning thyroid nodule classifier achieved an area under receiver operating characteristic curve (AUROC) of 0.889 on five-fold cross-validation. The AUROC improved when images were scaled by nodule size and six randomly selected cine clip frames were added to the training set per epoch. GradCAM class activation heatmaps revealed that microcalcifications and spongiform appearance were reliably recognized by the classifier as malignant and benign features, respectively. Spongiform nodules were found to be benign even when microcystic spaces constituted less than 50% of nodule volume.
To investigate the clinical relevance of the benign vs. malignant classifier, the binary classification threshold for the probability of malignancy generated by model was set at 7% to achieve sensitivity and negative predictive value (NPV) comparable to that of the fine needle aspiration biopsy (FNA). At this threshold, cross-validated deep-learning model achieved a sensitivity of 90%, specificity of 63%, positive predictive value (PPV) of 46% and negative predictive value of 94%. When tested on an independent image set that includes 18 classic papillary thyroid cancers (PTC), 5 follicular variant PTC, 4 medullary thyroid cancers, 3 follicular thyroid cancers (FTC), and 1 Hurthle cell thyroid cancer, the DL classifier achieved AUROC of 0.88, sensitivity of 97%, specificity of 61%, PPV of 40% and NPV of 99%. A single minimally-invasive FTC that had no suspicious features on thyroid ultrasound was incorrectly classified as benign.
Conclusions. This study demonstrates that the ultrasound-based deep-learning classifier of thyroid nodules achieves sensitivity and negative predictive value comparable to that of thyroid fine needle aspiration (FNA). Clinicians may use this tool to augment clinical judgment when determining whether to perform FNA procedures.

Deep learning analysis of thyroid nodule ultrasound images has high sensitivity and negative predictive value to rule-out thyroid cancer

Introduction
Hashimoto's thyroiditis raises the risk for differentiated thyroid cancer (DTC) in surgical series, but the role of thyroid peroxidase antibodies (TPO) remains controversial in that regard. We designed the present study to evaluate the effect of preoperative TPO titers in the risk of DTC.
Methods
A comprehensive search was conducted in PubMed, CENTRAL and Scopus databases in November 2021 for the terms "thyroid cancer" and "TPO" or "peroxidase antibodies". We characterized the differential risk found in patients with high titers of TPO (TPO+), as compared to those with low or undetectable titers (TPO-) with regard to DTC. Data are expressed as odds ratio (OR) with 95% confidence interval (CI).
Results
We retrieved and reviewed 408 records; 21 retrospective cohort studies (2006-2022) from 7 countries and n=30,5536 subjects, fulfilled the eligibility criteria: 15 studies in East Asia with n=25,125 subjects (82.2%) and 6 studies in the Western world with n=5,428 subjects (17.8%)]. These comprised n=17,684 subjects with benign disease (57.9%) and n=12,869 with DTC (42.1%). The reference used for TPO+ was 23.6±18.8 (Range 5.1-60.0 IU/ml); n=6,307 patients were TPO+ (20.6%) and n=24,246 were TPO- (79.4%). DTC was present in n=2,960 TPO+ patients (46.9%) and n=9,909 TPO- patients (40.9), OR 1.28, (95%CI 1.21-1.35), p<0.001.
A high titer of TPO was associated with increased risk for DTC in both Asian cohorts, [OR 1.36 (1.28-1.45, p<0.001)] and Western world alike, [OR 1.15 (1.02-1.31), p=0.025]. That risk was statistically significantly higher in the Asian world cohorts as compared to the Western World cohorts, p<0.001.

Conclusions
Thyroid peroxidase antibodies are an integral part of Hashimoto's thyroiditis, which is a well characterized risk factor for thyroid cancer and seem to increase that risk as well. That effect seems significantly higher in East Asia as compared to the Western World region. Further studies are needed to characterize the effects of the immune response, with regard to thyroid cancer risk.

The role of thyroid peroxidase antibodies on the risk of thyroid cancer: a systematic review and meta-analysis of surgical cohort studies.

Introduction: Radiofrequency ablation (RFA) is a promising, non-surgical method, used for the treatment of benign and malignant thyroid nodules (TN). While this minimally invasive technique has been studied extensively in Europe and Asia, it has been recently adopted in Latin America. The aim of our study is to describe the clinical outcomes following the first cohort of TN subjected to ablative radiofrequency in Ecuador.
Methods: This single-center, prospective study, carried out at ITECC (Instituto de la Tiroides y Enfermedades de Cabeza y Cuello) in Ecuador, included adults who underwent RFA for benign and malignant thyroid nodules (TN), between September 2020 and January 2022. The trans-isthmic approach and moving shot technique using the RF Medical equipment with an 18G needle and 5 or 7 mm tip were applied. Nodule size and volume, thyroid function, and complications were evaluated before and after RFA. Initial volume and change in TN volume were calculated with the ATA calculator and the independent-samples T-test.
Results: A total of 26 patients with 28 TNs were included. 25 TNs were benign (Bethesda II) and 3 were micropapillary thyroid cancer (MPTC) (Bethesda VI). In the benign lesions group, most were female (82.6%), <55 years of age (73.9%), euthyroid (62.5%), and symptomatic (95.6%); had a mean maximum diameter of 3.3 ± 1.3 (range: 0.8 – 5.5) cm and a mean initial volume of 13.1 ± 11.6 ml; and had predominantly solid component (36.4%). A substantial volume reduction was observed since the first month after RFA (1 month: mean volume 8.6 ± 4.2 ml, p=0.01; 6 months: mean volume 4.5 ± 3.9 ml, p=0.04; and 12 months: mean volume 4.9 ± 6.9 ml, p=0.25; vs. baseline). All patients were asymptomatic at the last follow-up. In the MPTC group, two patients were males (66.7%); all were <55 years of age (100.0%) and euthyroid (100.0%); most were asymptomatic diagnosed by routine US (66.7%); had a mean maximum diameter of 0.6 ± 0.1 (range: 0.5 – 0.8) cm and a mean initial volume of 0.056 ± 0.001 ml. At 1-month follow-up after RFA all TN were fibrosed and no lymph node or distant metastases were found. Finally, in both groups, no major complications were found and only one patient reported ecchymosis at the puncture site. Thyroid function was not affected.
Conclusions: To the best of our knowledge, we are the pioneers in Ecuador and the third country, after Brazil and Colombia, in Latin America that implemented RFA in the management of TN. In our first experience, RFA was a safe and effective technique. RFA induced a substantial volume reduction, improved symptoms, avoided cosmetic concerns, and did not affect normal thyroid function. Our results are consistent with other series. Data on a longer follow-up are needed to confirm long-term efficacy.

Radiofrequency Ablation for Thyroid Nodules: The First Ecuadorian Prospective Cohort

Background: The incidence of differentiated thyroid cancer (DTC) has tripled over the last 30 years in the US. While most patients respond to conventional treatment, some have an aggressive course with a dismal 5-year survival of 15.3%. There is a need to identify novel markers in addition to clinico-pathologic criteria to better prognosticate DTC patients. In addition, exploration of immunotherapies in advanced DTC underlies the critical need to investigate the immune interface of DTC to identify potential therapeutic targets and those likely to respond to immunotherapies.
Aims: We aimed to identify tumor-infiltrating immune cells and checkpoints [programmed cell death protein 1 (PD-1) and its ligand (PD-L1)] associated with DTC and investigate their association with metastatic and advanced DTC.
Methods: We performed multiplex immunofluorescence on de-paraffinized thyroid tissue collected at initial thyroidectomy from 17 adults with DTC. We identified cell-surface molecules to characterize the tumor immune microenvironment for leukocytes (CD45+), T cells (CD3+), B cells (CD19+), natural killer cells (CD56+), T regulatory cells (CD3+FOXP3+), CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), macrophages (CD68+), M2 macrophages (CD68+CD163+), and immune checkpoints PD-1 and PD-L1. Tonsil tissue was used as positive control. Immunofluorescence staining was quantified by cell counting HALO software. We compared the mean percentage expression of immune markers between tumor and adjacent thyroid tissue from the same patient by paired t-test, and in exploratory analysis between intra-tumoral tissue in patients with versus without distant metastases by student's t-test; p value<0.05 was used for significance.
Results: Overall, in 17 DTC patient samples, most leukocyte infiltrate occurred along the leading edge of the tumor and in the peritumoral thyroid tissue. Immune checkpoints PD-1 and PD-L1 were highly expressed in the tumor as compared to adjacent thyroid tissue (p<0.05). A higher trend for M2 macrophages was observed in the tumor but was not statistically significant. Intra-tumoral expressions of T regulatory cells, macrophages and PD-L1+ cells were more in samples for DTC patients with metastases (n=3) as compared to those without (n=14).
Conclusions: Immune profiling demonstrated significant differences between tumor and adjacent thyroid tissue, particularly for PD-1 and PD-L1, indicating their role in DTC occurrence. Intra-tumor infiltration of T regulatory cells, macrophages and PD-L1+ cells was more in metastatic DTC indicating their potential role in aggressive behavior of DTC. These data suggest that suppressive tumor immune microenvironment portends more aggressive behavior of DTC, and that specific immune suppressive markers may be potential therapeutic targets. We are continuing to expand our sample size to have adequate power for comparing groups with various clinico-pathologic severities and analyzing the role of pro-tumoral M2 macrophages in DTC prognosis.

Suppressive Tumor Immune Microenvironment is Associated with Aggressive Differentiated Thyroid Cancer

"Superhenhanced" transcription of specific oncogenes by aberrant looping of upstream enhancer elements to marked transcriptional regulatory regions is a mechanism of oncogene overexpression. Therefore, compounds that target key positive regulators of RNA polymerase II (RNAPII) activity, such as CDK7 and CDK9, can relatively selectively reduce expression of such superenhanced oncogenes and are in clinical trials. We have previously reported activity of the CDK7 inhibitor THZ1 in medullary thyroid cancer (MTC) along with identification of a superenhancer in RET. CDK7 inhibitors also are reported to be active in anaplastic thyroid cancer cell lines. In the present study, we extend this work to both CDK7 and CDK9 inhibitors, and to a more extensive set of thyroid cancer cell lines. We selected 8 thyroid cancer cell lines (TPC1, FTC133, BCPAP, SW1376; K1, THJ-16T, C643, and 8505C) from a variety of histological subtypes with different driver mutations for treatment with a CDK7 (THZ1) or CDK9 (AZD4573) inhibitors. Cell viability assays were performed to determine IC50 and Western blots were performed for target validation. IC50 values ranged from 5-100 nM for both compounds for all cell lines demonstrating broad activity and robust activity. Unexpectedly, both compounds inhibited both CDK7 RNAPII phosphorylation (Ser5) and CDK9 RNAPII phosphorylation (Ser2) due to a loss of total RNAPII protein levels in all cell lines. Further time course studies two cell lines, 8505C (BRAFV600E) and TPC1 (RET/PTC) showed the loss off RNAPII occurred rapidly (1-3 hours) with stable RNAPII mRNA levels by qRTPCR. We therefore hypothesized that RNAPII stalling induced by inhibition of CDK7 may cause loss of RNAPII protein stability through ubiquitination and proteasome-mediated degradation. Immunoprecipitation-Western blots demonstrated variable baseline levels of RNAPII ubiquitination in 8505C (low) and TPC1 (high) cells. THZ1 treatment induced ubiquitination of RNAPII in 8505C cells signficantly, while a modest increase beyond the higher baseline occurred in TPC1. Bortezomib (proteasome inhibitor) treatment rescued THZ1-reduced RNAPII protein levels in both cell lines. In summary, human thyroid cancer cell lines are broadly sensitive to CDK7 and CDK9 inhibitors. CDK 7 and 9 inhibitors reduce RNAPII levels and THZ1 induces RNAPII ubiquitination and proteasome degradation demonstrating a new mode of action for the reduced RNAPII activity induced by these inhibitors.

CDK7 and CDK9 Inhibition Induces RNA Polymerase II Ubiquitination and Proteasome Degradation in Thyroid Cancer Cells

Introduction: A well-known 'flip flop' phenomenon exists between whole-body scans (WBS) and 18FFDG PET imaging in differentiated thyroid cancer (DTC) such that cancer progression is typically associated with reduced iodine uptake and increased FDG avidity. Patients with FDG avidity have a poor clinical outcome while those with iodine avid lesions are more responsive to radioactive iodine therapy (RAI) and have a better prognosis. A spectrum however exists, as thyroid cancers become less well differentiated, when they will be both iodine and FDG avid. Current scoring systems in thyroid cancer are limited to a single imaging data set.  We propose a new scoring system (I-PET) combining WBS and FDG findings to identify patients more likely to be refractory to RAI, and who may be ideal candidates for 're-differentiation' before RAI treatment.
Methods: Retrospective analysis of 142 patients age > 18 with differentiated thyroid cancer who had a 18FFDG PET and WBS within a 6 month period of each other between 2010 and 2020. Initial pair of 18F FDG PET and WBS were reviewed by two independent nuclear medicine physicians and an IPET score was assigned: IPET [0]: Iodine –ve, FDG –ve, IPET [1]: Iodine +ve, FDG –ve, IPET [2]: Iodine +ve, FDG +ve and IPET [3]: Iodine –ve, FDG +ve. Patients with FDG +ve lesions (I-PET 2 and I-PET 3) were further classified into group 'a' and 'b' if SUVmax was less than or greater than 5 respectively. Follow-up data was obtained by chart review. Progression was defined as either structural recurrence as per RECIST 1.1 or equivalent clinical assessment, or biochemical incomplete response with an unstimulated Tg > 1ug/L. The study was approved by IRB (ETH00674/ STE01494).
Results: Of the 142 patients included in the study 121 patients had follow up data available for review. At baseline, 49 patients were classified as IPET [0], 10 as IPET [1], 16 as IPET [2] and 46 as IPET [3]. Progression was identified in 49% (59/121) patients; of which 54 were structural and 5 biochemical. Patients classified as [2b] and [3b] had a recurrence rate of 88% (7/8) and 78% (25/32) respectively, while IPET [0] patients had 22% (11/49) recurrence (p < 0.001). Patients classified as IPET [3b] were 10.4 times more likely to commence on tyrosine kinase inhibitor therapy (p=0.005) and had a 8.1 times greater mortality rate (p=0.02) than patients in the other IPET groups.
Conclusion: IPET score is a novel scoring system incorporating WBS and 18FFDG PET imaging which predicts progression and death in patients with FDG avid thyroid cancer.

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