Objective:The aim of the study was to evaluate the effect of health insurance status on rate of molecular testing (MT) and subsequent thyroidectomy in patients with indeterminate thyroid nodules in a real-world high-volume clinical setting. Methods:This is a single-center, retrospective study of patients who underwent fine-needle aspiration and had Bethesda III-IV cytology results between 2014 and 2022. We excluded patients with prior thyroid cancer, thyroidectomy, or incomplete insurance data. We utilized multivariable logistic regression to identify significant predictors of MT utilization and surgical intervention; specifically examining the role of insurance status and clinical symptoms. Results:A total of 448 patients were included (median age: 52 years, 79% women) with 79% of patients undergoing MT; with an associated 27-percentage-point (34% vs 61%, P < .001) reduction in thyroidectomy. Lack of MT was the strongest predictor of surgery (adjusted odds ratios [aOR]: 4.50, 95% confidence interval [CI]: 2.71-7.47, P < .001). However, uninsured/self-pay patients had 68% lower odds of MT (aOR: 0.32, 95% CI: 0.12-0.88, P = .026) and approximately 19-percentage-point higher thyroidectomy rate compared to insured patients (59% vs 40%). Thyroid-related compression symptoms were also significantly associated with increased rate of thyroidectomy (aOR: 1.86, P = .007) and an appropriate reduction in MT utilization (aOR: 0.62, P = .046). Conclusion:Despite high MT penetration at an academic medical center, lack of health insurance poses a critical barrier to evidence-based risk stratification leading to higher rates of diagnostic lobectomy. While symptoms appropriately drove surgical management, expanding affordable MT access may mitigate barriers to care for patients with thyroid nodules.

Background:Adrenocortical carcinoma (ACC) is a rare and aggressive endocrine cancer with limited treatment options and poor prognosis. Identifying novel therapeutic targets requires understanding the molecular drivers of ACC progression and establishing translational models for preclinical validation. Results:Matrix metalloproteinase-14 (MMP-14) is the most highly expressed MMP in ACC, and high MMP-14 expression is associated with worse overall and disease-free survival. We demonstrate that MMP-14 is essential for ACC cell survival and serves an unexpected role in maintaining genome stability. Genetic silencing or pharmacologic inhibition of MMP-14 significantly reduced viability in both NCI-H295R cells and patient-derived tumor organoids (PTOs). MMP-14 knockdown induced CHK1 activation and S-phase checkpoint arrest. Mechanistically, MMP-14 translocates to the nucleus and binds to chromatin following DNA damage induced by ionizing radiation or cisplatin. Loss of MMP-14 resulted in accumulation of DNA double-strand breaks, as evidenced by increased γH2AX foci, and impaired non-homologous end joining (NHEJ)-mediated repair. Conclusions:These findings reveal a novel nuclear function for MMP-14 in DNA repair and identify MMP-14 as a promising therapeutic target in ACC. Targeting MMP-14 may sensitize ACC tumors to DNA-damaging chemotherapy by impairing the repair of therapy-induced lesions.

Background: Medullary thyroid cancer (MTC) is a frequently metastatic tumor of the thyroid that develops from the malignant transformation of C-cells. These tumors most commonly have activating mutations within the RET or RAS proto-oncogenes. Germline mutations within RET result in C-cell hyperplasia, and cause the MTC pre-disposition disorder, multiple endocrine neoplasia, type 2A (MEN2A). Single-agent therapies for MTC, including vandetanib (VAN) and cabozantinib for all MTCs and selpercatinib (SEL) for RET-mutated MTC, lead to partial responses but are not curative.Methods: To identify new therapeutic targets for MTC, we conducted proteomic profiling of normal C-cells, MTC cells, pre-malignant MEN2A patient samples, and MTC tumors.Results: From this analysis, we identified CAPN1, a member of the CALPAIN (CAPN) family endopeptidases, as widely upregulated in MTC samples. We found that short hairpin RNA-mediated depletion of CAPN1 or inhibitors of CAPN1 significantly reduced MTC cell growth, colony formation, and xenograft tumor growth in vivo. In addition, we show that CAPN1 inhibitors synergize with VAN and SEL in vitro, maximizing apoptosis. Mechanistic experiments implicate CAPN1 in inhibiting neurofibromin, encoded by NF1, and CAPN1 inhibitors stabilize NF1 protein levels and diminish downstream RAS/RET activation of AKT and ERK.Conclusions: Our data suggest that increased CAPN1 levels support RET and RAS-fueled growth by reducing NF1 levels. We find that combinatorial therapies between CAPN1 inhibitors and VAN or SEL show maximal efficacy in MTC cells.

BACKGROUND:How aldosterone synthase (CYP11B2) staining patterns impact patient outcomes in those with unilateral primary aldosteronism is not well described. We hypothesized that a system of categorization would benefit future research and that clinical and biochemical outcomes after unilateral adrenalectomy are impacted by different CYP11B2 staining patterns. METHODS:A retrospective review of patients undergoing adrenalectomy for primary aldosteronism from January 2015 to September 2023 was conducted. Demographics, clinical, and pathologic data were analyzed. A system of categorization of staining patterns was developed. Clinical and biochemical outcomes were compared with staining patterns to assess differences and determine correlation. Descriptive and statistical analyses were performed using SPSS. RESULTS:Forty-three patients were included. The following CYP11B2 staining patterns were identified: (1) single adenoma; (2) aldosterone producing nodule(s) or micronodule(s); (3) combination of type 1 and type 2; (4) hyperplasia; and (5) aldosterone-producing adrenocortical cancer. In total, 23 of 43 revealed CYP11B2 staining in a single adenoma only. Staining in 3/23 involved a portion of the adenoma. 4/9 patients age <40 had areas of CYP11B2 staining in nonadenomatous tissue. Complete biochemical cure was noted in 37 of 43 (86%) and complete clinical cure in 23.2%. There were no differences between staining pattern and sex, race, or age. CYP11B2 staining pattern did not correlate with early clinical or biochemical outcomes. CONCLUSION:Adrenalectomy specimens from patients treated for primary aldosteronism reveal multiple CYP11B2 staining patterns, including in nonadenomatous tissue in many patients. The impact of these patterns on clinical outcomes requires additional investigation. Uniform categorization of staining patterns will allow for consistent reporting across studies.

INTRODUCTION Our university-based surgery department recently transitioned to attending-only authorship of operative reports. We performed a mixed-methods investigation to determine if trainee-initiated endocrine surgical reports were associated with under-coding of specific procedures. METHODS Endocrine operations performed from July 2020 to June 2022 were identified from billing data. Pre- and post-policy RVU distributions and note modification history were reviewed to determine how often trainees captured billable differentiators over attending note modification. RESULTS 714 operations and 1138 billed procedures were identified. Parathyroidectomy alone showed greater mean RVUs with attending-only reports attributable to attending practice change in coding for intraoperative parathyroid hormone monitoring. Trainees were more likely to miss coding modifier 22 but RVU losses were prevented by attending note modification. CONCLUSION Trainee-initiated operative reports were not associated with RVU losses for endocrine operations compared to attending-only reports. Trainee dictation can be improved by emphasizing education on procedural billing differences and surgical reasoning.

BackgroundAlthough uncommon, adrenal hemorrhage has multiple etiologies. Because clinical characteristics, management, and outcomes of patients with adrenal hemorrhage are inadequately described, we examined the underlying etiology, need for intervention, evolution of imaging characteristics, and adequacy of subsequent evaluation.MethodsWe performed a retrospective review of patients diagnosed with adrenal hemorrhage (radiologist-confirmed density consistent with hemorrhage on computed tomography) from 2005 to 2021 at a university-based institution. Demographic characteristics, hemorrhage etiology, and subsequent follow-up were analyzed.ResultsOf 193 adrenal hemorrhage patients, the mean age was 49.2 ± 18.3 years, and 35% were female. Clinical presentations included trauma (47%), abdominal or flank pain (28%), incidental findings on imaging acquired for other reasons (12%), postoperative complication (8%), or shock (3%). Hemorrhage outside of the gland was present in 62% of patients. Unilateral hemorrhage was more frequent (93%) than bilateral (7%). A total of 12% of patients had nodules, but only 70% of these were identified on initial imaging, and only 43% had hormonal evaluation. Of 7 patients who had adrenalectomy or biopsy, pathology was either benign (57%) or nonadrenal malignancy (43%). No adrenocortical carcinomas were identified. Follow-up imaging was performed in 56% of patients and revealed decreased, stable, resolved, or increased adrenal hemorrhage size in 39%, 19%, 30%, and 12% of patients, respectively.ConclusionAdrenal hemorrhage is secondary to multiple etiologies, most commonly trauma. In the setting of adrenal hemorrhage, many adrenal nodules were not identified on initial imaging. Only a minority of patients with nodules underwent “complete” biochemical evaluation. Follow-up imaging may improve the identification of underlying nodules needing hormonal evaluation.

BACKGROUND:Thoracic duct leaks occur in up to 5% of left lateral neck dissections. No one imaging modality is routinely used to identify the thoracic duct intraoperatively. The goal of our study was to evaluate the efficacy and safety of indocyanine green lymphangiography for intraoperative identification of the thoracic duct compared to traditional methods using ambient and evaluate the optimal timing of indocyanine green administration.METHODS:We enrolled all patients who underwent left lateral neck dissection at our institution from 2018 to 2022 in this prospective clinical trial. After indocyanine green injection into the dorsum of the foot, we performed intraoperative imaging was performed with a near-infrared fluorescence camera. We reported the data using descriptive statistics.RESULTS:Of the 42 patients we enrolled, 14 had prior neck surgery, and 3 had prior external beam radiation. We visualized the thoracic duct with ambient light in 48% of patients and with near-infrared fluorescence visualization in 64%. In 17% of patients, we could identify the thoracic duct only using near-infrared fluorescence visualization, which occurred within 3 minutes of injection, and were required to re-dose 5 patients. We visualized the thoracic duct with near-infrared fluorescence in all patients with prior neck radiation and 77% of patients with prior neck surgery. One adverse reaction occurred (hypotension), and 5 intraoperative thoracic duct injuries occurred that were ligated. There with no chylous fistulas postoperatively.CONCLUSION:This trial demonstrates that near-infrared fluorescence identification of the thoracic duct is feasible and safe with indocyanine green lymphangiography, even in patients with prior neck surgery or radiation.

Malignant struma ovarii is an exceedingly rare pathology with a paucity of established criteria regarding management and surveillance with recommendations largely based on case reports and retrospective data. Many authors have supported stratification of malignant struma ovarii into low vs high-risk disease with more conservative management reserved for those deemed low-risk. Here we present a unique case of recurrent metastatic malignant struma ovarii after surveillance was undertaken in the setting of initially low-risk disease.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with a poor prognosis and limited treatment options. Bulk genomic characterization of ACC has not yielded obvious therapeutic or immunotherapeutic targets, yet novel therapies are needed. We hypothesized that elucidating the intratumoral cellular heterogeneity by single nuclei RNA sequencing analyses would yield insights into potential therapeutic vulnerabilities of this disease. In addition to characterizing the immune cell and fibroblast landscape, our analyses of single nuclei gene expression profiles identified an adrenal cortex cell cluster exhibiting a program of replication stress and DNA damage response in primary and metastatic ACC. In vitro assessment of replication stress and DNA damage response using an ACC cell line and a series of newly-derived hormonally active patient-derived tumor organoids revealed ATR sensitivity. These findings provide novel mechanistic insight into ACC biology and suggest that an underlying dependency on ATR may be leveraged therapeutically in advanced ACC.

BACKGROUND:Primary adenoma (PA) and multi-gland hyperplasia (MGH) account for 85% and 15% of primary hyperparathyroidism (PHPT) cases, respectively. Near-infrared autofluorescence (NIRAF) enhances intraoperative parathyroid identification. We hypothesized that PA would display a more heterogeneous NIRAF pattern compared to MGH. METHODS:Patients undergoing surgery for sporadic PHPT were categorized based on the presence of PA or MGH. To quantify heterogeneity, we utilized ratios of (1) mean parathyroid gland (PG) NIRAF over background NIRAF (mean ratio), (2) minimum and (3) maximum PG NIRAF over mean PG NIRAF (minimum and maximum ratios). Additionally, a heterogeneity score was quantified using mean ratio (mean PG NIRAF over background NIRAF), and overall NIRAF (mean NIRAF of eight random 15 × 15 pixel areas). A point was assigned to ratios <0.8 or >1.2. Images were quantified by ImageJ software. Mann-Whitney test was performed for all comparisons. RESULTS:Of 78 patients, 63 had a single PA and 15 had MGH, totaling 102 PGs. There was no difference between their mean ratios. PA had a lower minimum ratio compared to that of MGH (0.86 ± 0.01 vs. 0.93 ± 0.01, p = 0.001) and a brighter maximum ratio (1.21 ± 0.02 vs. 1.12 ± 0.01, p = 0.0008). PA also scored higher on their heterogeneity scores compared to MGH (1.27 ± 0.23 vs. 0.33 ± 0.15, p = 0.001). CONCLUSION:Single parathyroid adenomas display a more heterogeneous autofluorescence pattern compared to that of multi-gland hyperplasia. Intraoperative characterization of PGs by real-time NIR imaging patterns may be a beneficial adjunct during parathyroid surgery.

The reported threshold of a near-infrared fluorescence detection probe (FDP) for judging parathyroid glands (PGs) is based on the autofluorescence intensity relative to other non-PG tissues, making it unreliable when not enough reference tissues are measured. We aim to convert FDP into a more convenient tool for identifying accidentally resected PGs by quantitative measurements of autofluorescence in resected tissues. It was a prospective study approved by the Institutional Review Board. The research was divided into two stages: (1) In order to calibrate the novel FDP system, autofluorescence intensity of different in / ex vivo tissues was measured and the optimal threshold was obtained using receiver operating characteristic (ROC) curve. (2) To further validate the effectiveness of the new system, detection rates of incidental resected PGs by pathology in the control group and by FDP in the experimental group were compared. Autofluorescence of PGs was significantly higher than that of non-PG tissue (43 patients, Mann–Whitney U test, p < 0.0001). An optimal threshold of sensitivity / specificity (78.8

Objectives: To describe the first reported use of neoadjuvant dabrafenib and trametinib specifically to permit organ conservation surgery in locally advanced recurrent differentiated thyroid carcinoma.Patients and methods: A patient presented with locally recurrent, radioiodide-resistant DTC with a BRAF V600E mutation invading the laryngotrachea. Definitive treatment would require a total laryngectomy. She was offered neoadjuvant dabrafenib and trametinib prior to surgery.Results: A significant radiographic response permitted partial laryngectomy, enabling preservation of voice, early resumption of oral feeding, and avoidance of permanent tracheostomy. At 9 months, she remained free of disease.Conclusion: Neoadjuvant tyrosine kinase inhibitor treatment prior to definitive surgery for locally-invasive recurrent DTC is a potential approach that may limit the degree of surgery and associated morbidity.

Background: In recent years, fluorescence imaging-relying both on parathyroid gland autofluorescence under near-infrared light and angiography using the fluorescent dye indocyanine green-has been used to reduce risk of iatrogenic parathyroid injury during thyroid and parathyroid resections, but no published guidelines exist regarding its use. In this study, orchestrated by the International Society for Fluorescence Guided Surgery, areas of consensus and nonconsensus were examined among international experts to facilitate future drafting of such guidelines. Methods: A 2-round, online Delphi surveywas conducted of 10 international experts in fluorescence imaging use during endocrine surgery, asking them to vote on 75 statements divided into 5 modules: 1 = patient preparation and contraindications to fluorescence imaging (n = 11 statements); 2 = technical logistics (n = 16); 3 = indications (n = 21); 4 = potential advantages and disadvantages of fluorescence imaging (n = 20); and 5 = training and research (n = 7). Several methodological steps were taken to minimize voter bias. Results: Overall, parathyroid autofluorescence was considered better than indocyanine green angiography for localizing parathyroid glands, whereas indocyanine green angiography was deemed superior assessing parathyroid perfusion. Additional surgical scenarios where indocyanine green angiography was thought to facilitate surgery are (1) when >1 parathyroid gland requires resection; (2) during redo surgeries, (3) facilitating parathyroid autoimplantation; and (4) for the predissection visualization of abnormal glands. Both parathyroid autofluorescence and indocyanine green angiography can be used during the same procedure and employing the same imaging equipment. However, further research is needed to optimize the dose and timing of indocyanine green administration. Conclusion: Though further research remains necessary, using fluorescence imaging appears to have uses during thyroid and parathyroid surgery. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction: Parathyroid carcinoma is very rare, and intraoperative definitive diagnosis can be elusive with currently available diagnostics. Near-infrared (NIR) autofluorescence is an emerging tool that identifies parathyroid glands in real time. It is not known whether NIR autofluorescence can detect parathyroid carcinoma intraoperatively. Methods: Patients with preoperative suspicion for parathyroid carcinoma were identified from ongoing studies examining parathyroid autofluorescence with a NIR camera and probe. Specimens from these patients were examined intraoperatively to determine their autofluorescence patterns. Results: Three patients with suspected parathyroid carcinoma were identified preoperatively. Intraoperative NIR autofluorescence imaging showed a relative lack of autofluorescence for all cases, in contrast to parathyroid adenomas and normal parathyroid glands, which typically exhibit significant autofluorescence. Final pathology confirmed parathyroid carcinoma in all cases. Conclusion: Parathyroid carcinoma can be difficult to confirm prior to final pathology review. Our 3 cases suggest that absence of NIR autofluorescence may suggest the likelihood of parathyroid carcinoma, but more studies are needed to investigate this experience.

ObjectivesTo evaluate the frequency and risk of malignancy of TSHRpI568T mutations discovered in indeterminate thyroid nodules (ITN) within the Veracyte CLIA laboratory undergoing Afirma® Genomic Sequencing Classifier (GSC) testing, and to evaluate a broader cohort of TSHR variants and their categorization as Afirma GSC benign (GSC-B) or suspicious (GSC-S). Finally, we seek to assess the risk of malignancy (ROM) of this group of TSHR mutated ITN in the GSC-S category.MethodsITN submitted to Veracyte for Afirma GSC testing between October 2017 and February 2022 were analyzed for TSHR variants and rates of GSC-B and GSC-S were calculated based upon BIII or IV cytology, by TSHR variant codon amino acid (AA) substitution, age, and gender. For GSC-S samples, surgical pathology reports were requested, and the rate of malignancy was calculated.ResultsFive percent of the ITN samples harbored an isolated TSHR variant and 5% of those were classified as GSC-S. Among TSHRpI568T samples, 96% were GSC-B and of the GSC-S samples, 21% were malignant. Among an unselected group of TSHR, absent TSHRpI568T mutations, 16.3% of GSC-S samples were malignant, all but one with codon mutations in the transmembrane subdomains of the TSHR. This prompted a dedicated evaluation of transmembrane codons which revealed a malignancy rate of 10.7% among GSC-S nodules. In total, 13/85 (15.3%) TSHR mutated ITN with Afirma GSC-S results were found to be malignant.ConclusionsTSHR variants are rare in ITN, and most are categorized as benign under Afirma GSC testing which carries a < 4% risk of malignancy. For GSC-S ITN with TSHR mutations, the risk of malignancy is ≥= 15%, which is clinically meaningful and may alter treatment or monitoring recommendations for patients.

INTRODUCTION: Primary adenoma (PA) and multigland hyperplasia (MGH) account for 85% and 15% of primary hyperparathyroidism (pHPT) cases, respectively. Near-infrared autofluorescence (NIRAF) enhances intraoperative parathyroid identification. We hypothesized that PA would display a more heterogeneous NIRAF pattern than MGH. METHODS: Patients undergoing surgery for sporadic pHPT were categorized based on the presence of PA or MGH. To quantify heterogeneity, we used ratios of mean parathyroid gland (PG) NIRAF over background NIRAF (mean ratio), minimum and maximum PG NIRAF over mean PG NIRAF (minimum and maximum ratios). Additionally, a heterogeneity score was quantified using the mean ratio (mean PG NIRAF over background NIRAF), and overall NIRAF (mean NIRAF of 8 random 15×15 pixel areas). A point was assigned to ratios <0.8 or >1.2. Images were quantified by ImageJ software. Mann-Whitney test was performed for all comparisons. RESULTS: Of 78 patients, 63 had a single PA and 15 had MGH, totaling 102 PGs. There was no difference between their mean ratios. PA had a lower minimum ratio than that of MGH (0.86 ± 0.01 vs 0.93 ± 0.01, p = 0.001) and a brighter maximum ratio (1.21 ± 0.02 vs 1.12 ± 0.01, p = 0.0008). PA also scored higher on their heterogeneity scores than MGH (1.27 ± 0.23 vs 0.33 ± 0.15, p = 0.001). In sum, PA displayed a more heterogeneous pattern with significantly brighter and darker areas (Figure).FigureCONCLUSION: Single parathyroid adenomas display a more heterogeneous autofluorescence pattern compared with that of MGH. Intraoperative characterization of PGs by real-time near-infrared imaging patterns may be a beneficial adjunct during parathyroid surgery.

Context: Parathyroid tissue is one of the few tissues to have strong near-infrared (NIR) autofluorescence, which has been exploited to improve intraoperative parathyroid identification. The US Food and Drug Administration has approved 2 devices for this purpose. Adrenal glands can be difficult to distinguish from surrounding fat, an issue during total adrenalectomy. Objective: We hypothesized adrenal tissue may also possess considerable NIR autofluorescence. Methods: Resected patient adrenal specimens were examined after robotic adrenalectomy with an NIR camera intraoperatively. Patients did not receive fluorescent dye. Images were taken of both gross and sectioned specimens. Post hoc image analysis was performed with ImageJ software. Confocal microscopy was performed on selected tissues using immunofluorescence and hematoxylin-eosin staining. Results: Resected tissue was examined from 22 patients undergoing surgery for pheochromocytomas (6), primary aldosteronism (3), adrenocorticotropin-independent hypercortisolism (10), and a growing or suspicious mass (3). Normal adrenal tissue demonstrated strong NIR autofluorescence. The intensity ratio compared to background (set as 1) for gross images was 2.03 +/- 0.51 (P < .0001) compared to adjacent adipose of 1.24 +/- 0.18. Autofluorescence from adrenal tumors was also detected at variable levels of intensity. Cortisol-producing tumors had the highest fluorescence ratio of 3.01 +/- 0.41. Confocal imaging localized autofluorescence to the cytosol, with the highest intensity in the zona reticularis followed by the zona fasciculata. Conclusion: Normal and abnormal adrenal tissues possess natural NIR autofluorescence. Highest autofluorescence levels were associated with cortisol-producing tumors. Confocal imaging demonstrated the highest intensity in the zona reticularis. NIR cameras may have the potential to improve identification of adrenal tissue during surgery.

Background: Intraoperative parathyroid gland identification can be challenging. Parathyroid glands have an intrinsic autofluorescence when excited by wavelengths in the near-infrared region. Studies using near-infrared cameras to detect parathyroid gland near-infrared autofluorescence have suggested improved identification. The pathologic parathyroid glands in primary hyperparathyroidism have variable near-infrared autofluorescence intensity, but how this correlates with different characteristics of hyperparathyroidism is unknown. Our objective was to correlate the fluorescent intensity of excited glands with clinical variables to enhance a surgeon's ability to identify parathyroid glands. Methods: The data on patients undergoing surgery for primary hyperparathyroidism were collected. The images were collected intraoperatively with a handheld near-infrared device and analyzed. The data consisted of the ratio of mean parathyroid gland near-infrared autofluorescence over background (white gauze) near-infrared autofluorescence. The variables assessed for correlation with autofluorescence intensity were gland volume and weight, preoperative serum calcium and parathyroid hormone, age, body mass index, and sex. The images were quantified by Image J software (National Institutes of Health, Bethesda, MD). The lasso regression was analyzed by R version 4.1.3 to calculate adjusted P values (R Foundation for Statistical Computing, Vienna, Austria). Results: From 2017 to 2021, 131 patients with primary hyperparathyroidism underwent parathyroidectomies of 151 parathyroid glands. The mean near-infrared autofluorescence intensity of parathyroid glands had a negative correlation with weight with lighter glands fluorescing more (P =.019) and a positive correlation with age with glands from older patients fluorescing more (P =.013). There were no significant correlations with preoperative serum calcium and parathyroid hormone, body mass index, and sex (P >.05). Conclusion: In patients with primary hyperparathyroidism, we found that autofluorescence intensity correlated with parathyroid gland weight and patient age. This suggested that near-infrared camera use may be particularly helpful in identifying smaller adenomas and in older patients. (c) 2022 Elsevier Inc. All rights reserved.

Abstract Background 18 F‐Fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) positive (PET+) cytologically indeterminate thyroid nodules (ITNs) have variable cancer risk in the literature. The benign call rate (BCR) of Afirma Gene Classifier (Gene Expression Classifier, GEC, or Genome Sequence Classifier, GSC) in (PET +) ITNs is unknown. Methods This is a retrospective study at our institution of all patients with (PET+) ITNs (Bethesda III/IV) from 1 January 2010 to 21 May 2019 who underwent Afirma testing and/or surgery or repeat FNA with benign cytology. Results Forty‐five (PET+) ITNs were identified: 31 Afirma‐tested (GEC = 20, GSC = 11) and 14 either underwent surgery (n = 13) or repeat FNA (Benign cytology) (n = 1) without Afirma. The prevalence of cancer and noninvasive follicular thyroid neoplasm with papillary‐like nuclear features (NIFTP) including only resected nodules and ITN with repeat benign FNA (n = 33) was 36.4% (12/33). Excluding all Afirma “suspicious” non‐resected ITNs and assuming all Afirma “benign” ITNs were truly benign, that prevalence was 28.6% (12/42). The BCR with GSC was 64% compared to 25% with GEC (p = 0.056). Combining GSC/GEC‐tested ITNs, the BCR was higher in ITNs demonstrating low/very low‐risk sonographic pattern by the American Thyroid Association (ATA) classification and ITNs scoring <4 by the American College of Radiology Thyroid Imaging, Reporting and Data System (ACR‐TI‐RADS) than ITNs with higher sonographic pattern/score (p = 0.025). Conclusions The prevalence of cancer/NIFTP in (PET+) ITNs was 28.6–36.4% depending on the method of calculation. The BCR of Afirma GSC was 64%. Combining Afirma GEC/GSC‐tested ITNs, BCR was higher in ITNs with a lower risk sonographic pattern.