Proposal for standardized ultrasound analysis of the salivary glands: Part 1 submandibular gland.

Objectives: The Salivary Gland Committee of the American Academy of Otolaryngology-Head and Neck Surgery seeks to standardize terminology and technique for ultrasonograpy used in the evaluation and treatment of salivary gland disorders. Methods: Development of expert opinion obtained through interaction with international practitioners representing multiple specialties. This committee work includes a comprehensive literature review with presentation of case examples to propose a standardized protocol for the language used in ultrasound salivary gland assessment. Results: A multiple segment proposal is initiated with this focus on the submandibular gland. We provide a concise rationale for recommended descriptive language highlighted by a more extensive supplement that includes an extensive literature review with additional case examples. Conclusion: Recommendations are provided to improve consistency both in performing and reporting submandibular gland ultrasound.

Novel Multidisciplinary Salivary Gland Society (MSGS) Questionnaire: An International Consensus.

OBJECTIVES: First, establishment and validation of a novel questionnaire documenting the burden of xerostomia and sialadenitis symptoms, including quality of life. Second, to compare two versions regarding the answering scale (proposed developed answers Q3 vs. 0-10 visual analogue scale Q10) of our newly developed questionnaire, in order to evaluate their comprehension by patients and their reproducibility in time. STUDY DESIGN: The study is a systematic review regarding the evaluation of the existing questionnaire and a cohort study regarding the validation of our new MSGS questionnaire. MATERIALS AND METHODS: A Multidisciplinary Salivary Gland Society (MSGS) questionnaire consisting of 20 questions and two scoring systems was developed to quantify symptoms of dry mouth and sialadenitis. Validation of the questionnaire was carried out on 199 patients with salivary pathologies (digestive, nasal, or age-related xerostomia, post radiation therapy, post radioiodine therapy, Sjögren's syndrome, IgG4 disease, recurrent juvenile parotitis, stones, and strictures) and a control group of 66 healthy volunteers. The coherence of the questionnaire's items, its reliability to distinguish patients from healthy volunteers, its comparison with unstimulated sialometry, and the time to fill both versions were assessed. RESULTS: The novel MSGS questionnaire showed good internal coherence of the items, indicating its pertinence: the scale reliability coefficients amounted to a Cronbach's alpha of 0.92 for Q10 and 0.90 for Q3. The time to complete Q3 and Q10 amounted, respectively, to 5.23 min (±2.3 min) and 5.65 min (±2.64 min) for patients and to 3.94 min (±3.94 min) and 3.75 min (±2.11 min) for healthy volunteers. The difference between Q3 and Q10 was not significant. CONCLUSION: We present a novel self-administered questionnaire quantifying xerostomia and non-tumoral salivary gland pathologies. We recommend the use of the Q10 version, as its scale type is well known in the literature and it translation for international use will be more accurate. Laryngoscope, 132:322-331, 2022.

Immunosuppressed Miniswine as a Model for Testing Cell Therapy Success: Experience With Implants of Human Salivary Stem/Progenitor Cell Constructs.

An urgent need exists to develop large animal models for preclinical testing of new cell therapies designed to replace lost or damaged tissues. Patients receiving irradiation for treatment of head and neck cancers frequently develop xerostomia/dry mouth, a condition that could one day be treated by cell therapy to repopulate functional saliva-producing cells. Using immunosuppression protocols developed for patients receiving whole face transplants, we successfully used immunosuppressed miniswine as a suitable host animal to evaluate the long-term stability, biocompatibility, and fate of matrix-modified hyaluronate (HA) hydrogel/bioscaffold materials containing encapsulated salivary human stem/progenitor cells (hS/PCs). An initial biocompatibility test was conducted in parotids of untreated miniswine. Subsequent experiments using hS/PC-laden hydrogels were performed in animals, beginning an immunosuppression regimen on the day of surgery. Implant sites included the kidney capsule for viability testing and the parotid gland for biointegration time periods up to eight weeks. No transplant rejection was seen in any animal assessed by analysis of the tissues near the site of the implants. First-generation implants containing only cells in hydrogel proved difficult to handle in the surgical suite and were modified to adhere to a porcine small intestinal submucosa (SIS) membrane for improved handling and could be delivered through the da Vinci surgical system. Several different surgical techniques were assessed using the second-generation 3D-salivary tissue (3D-ST) for ease and stability both on the kidney capsule and in the capsule-less parotid gland. For the kidney, sliding the implant under the capsule membrane and quick stitching proved superior to other methods. For the parotid gland, creation of a tissue "pocket" for placement and immediate multilayer tissue closure were well tolerated with minimal tissue damage. Surgical clips were placed as fiduciary markers for tissue harvest. Some implant experiments were conducted with miniswine 90 days post-irradiation when salivation decreased significantly. Sufficient parotid tissue remained to allow implant placement, and animals tolerated immunosuppression. In all experiments, viability of implanted hS/PCs was high with clear signs of both vascular and nervous system integration in the parotid implants. We thus conclude that the immunosuppressed miniswine is a high-value emerging model for testing human implants prior to first-in-human trials.

Salivary gland disease in the era of COVID-19 pandemic.

Coronavirus disease 2019 (COVID-19) pandemic forced significant changes in current approach to outpatient evaluation of common otolaryngology complaints as hospitals around the world are trying to limit the spread of the virus and to preserve health care resources. These changes raise a lot of questions regarding patient triage and treatment decisions in clinical situations when it is unclear if the workup and management can be postponed. In this communication, we present our approach to evaluation and triage of new patients with complaints concerning for salivary gland disease.

Dynamic Assembly of Human Salivary Stem/Progenitor Microstructures Requires Coordinated α1ß1 Integrin-Mediated Motility.

A tissue engineering approach can provide replacement salivary gland structures to patients with hyposalivation disorders and xerostomia. Salivary human stem/progenitor cells (hS/PCs) were isolated from healthy regions of parotid glands of head and neck surgery patients, expanded, then encapsulated in biocompatible hyaluronate (HA)-based hydrogels. These bioactive hydrogels provide a surrogate territorial matrix suitable for the dynamic assembly, growth and reorganization of salivary gland components. This study examined the dynamics of salivary microstructure formation, growth, and reorganization using time-lapse imaging over 15 h. Immunofluorescence detection monitored production of individual basement membrane components forming around developing microstructures, and Ki67 assessed proliferation. Dynamic movements in hydrogels were quantified by measuring angular velocity (ω) of rotating salivary microstructures and changes in basement membrane architecture during microstructure growth. Integrin involvement in the dynamic reassembly was assessed using knockdown and inhibitor approaches. Single hS/PCs expanded over 5 days into spherical microstructures typically containing 3-10 cells. In larger macrostructures, proliferation occurred near the peripheral basement membrane that underwent growth-associated cycles of thinning and collapse. De novo secretion of laminin/collagen IV from reorganizing hS/PCs preceded that of perlecan/HSPG2. Microstructures routinely expressed ß1 integrin-containing complexes at basement membrane-associated regions and exhibited spontaneous and coordinated rotation during basement membrane maturation. ß1 integrin siRNA knockdown at the single-cell state prevented hS/PC microstructure growth. After microstructure formation, ß1 integrin knockdown reduced rotation and mean ω by 84%. Blockade of the α1 integrin subunit (CD49a) that associates with ß1 reduced mean ω by 66%. Studies presented here show that initial hS/PC structure growth and basement membrane maturation depends on α1ß1-integrin mediated signaling. Coordinated cellular motility during neotissue reorganization reminiscent of salivary gland acini was critically dependent both on hS/PC-secretion of laminin,collagen type-IV, and perlecan/HSPG2 and the force-driven interactions of α1ß1-integrin activation. We conclude that α1ß1-integrin plays a critical role in establishing human salivary gland coordinated structure and function, and that its activation in tissue engineered systems is essential to tissue assembly.

Serial patient-derived orthotopic xenografting of adenoid cystic carcinomas recapitulates stable expression of phenotypic alterations and innervation.

BACKGROUND: Patient-derived xenograft (PDX) models have significantly enhanced cancer research, and often serve as a robust model. However, enhanced growth rate and altered pathological phenotype with serial passages have repeatedly been shown in adenoid cystic carcinoma (ACC) PDX tumors, which is a major concern. METHODS: We evaluated the fidelity of ACCs in their natural habitat by performing ACC orthotopic xenotransplantation (PDOX) in salivary glands. FINDINGS: Our PDOX model enabled solid tumors to integrate within the local epithelial, stromal and neuronal environment. Over serial passages, PDOX tumors maintained their stereotypic MYB-NFIB translocation, and FGFR2 and ATM point mutations. Tumor growth rate and histopathology were retained, including ACCs hallmark presentations of cribriform, tubular, solid areas and innervation. We also demonstrate that the PDOX model retains its capacity as a tool for drug testing. INTERPRETATION: Unlike the precedent PDX model, our data shows that the PDOX is a superior model for future cancer biology and therapy research. FUND: This work was supported by the National Institutes of Health (NIH)/National Institute of Dental and Craniofacial Research (NIDCR) grants DE022557, DE027034, and DE027551.

Combination of post-operative radiotherapy and cetuximab for high-risk cutaneous squamous cell cancer of the head and neck: A propensity score analysis.

OBJECTIVES: The objective of this study was to investigate the safety, tolerability and preliminary efficacy of radiotherapy plus cetuximab in high risk CSCC patients. MATERIALS AND METHODS: Patients with high-risk CSCC diagnosed between 2006 and 2013 were analyzed. Patients were divided into two groups: radiotherapy alone versus radiotherapy plus cetuximab. Among 68 patients meeting study criteria, we identified 29 treated with cetuximab plus RT and 39 with RT alone. Primary analysis examined disease-free and overall survival, freedom from local and distant recurrence in the propensity score matched cohort. Propensity score analysis was performed with weighted factors including: Charlson Comorbidity Index score, age. KPS, primary location, T and N stage, recurrent status, margin status, LVSI, PNI and grade. Toxicity was assessed using the CTCAE v4.0. RESULTS: Median follow-up for living patients was 30 months. Patients in the cetuximab group were more likely to have advanced N stage, positive margins and recurrent disease. After propensity score matching the groups were well balanced. Six patients experienced ≥ grade 3 acute toxicity in the cetuximab group. The 1-year, 2-year and 5-year progression free survival (PFS) for patients in the cetuximab group were 86%, 72% and 66%, respectively. The 1-year, 2-year and 5-year overall survival (OS) for patients in the cetuximab group was 98%, 80% and 80%, respectively. CONCLUSIONS: Although limited by small numbers, the combination of cetuximab and radiotherapy in CSCC appears well tolerated there were more long-term survivors and less distant metastasis in the cetuximab group. These promising finding warrant further studies.

Molecular testing for thyroid nodules: Review and current state.

Thyroid nodules affect nearly two-thirds of the world population. Fine-needle biopsy with cytologic evaluation remains the diagnostic test of choice to distinguish benign from malignant thyroid nodules yet fails to discriminate as benign or malignant in up to one-third of cases. This review discusses the limitation of current cytopathologic evaluation, the development of thyroid molecular testing, and the strengths and limitations of commercially available tests. Initial cytomolecular testing sought to identify specific gene mutations associated with thyroid cancer. Although the presence of a mutation was strongly associated with cancer, the likelihood of identifying a mutation was low; therefore, the test had low sensitivity. Subsequent tests developed have sought to improve the accuracy of cytomolecular testing for thyroid fine-needle aspirations, both to reassure patients and providers when malignancy may be absent and to confirm the malignancy when present. The development of cytomolecular testing for thyroid nodules has informed and improved current understanding of thyroid nodule formation and progression. When used appropriately and with clear understanding of the advantages and disadvantages, cytomolecular testing has the potential to improve patient care in the setting of indeterminate thyroid nodules by helping to guide both the need for and the extent of thyroid surgery. Cancer 2018;124:888-98. © 2017 American Cancer Society.

Bottom-up assembly of salivary gland microtissues for assessing myoepithelial cell function.

Myoepithelial cells are flat, stellate cells present in exocrine tissues including the salivary glands. While myoepithelial cells have been studied extensively in mammary and lacrimal gland tissues, less is known of the function of myoepithelial cells derived from human salivary glands. Several groups have isolated tumorigenic myoepithelial cells from cancer specimens, however, only one report has demonstrated isolation of normal human salivary myoepithelial cells needed for use in salivary gland tissue engineering applications. Establishing a functional organoid model consisting of myoepithelial and secretory acinar cells is therefore necessary for understanding the coordinated action of these two cell types in unidirectional fluid secretion. Here, we developed a bottom-up approach for generating salivary gland microtissues using primary human salivary myoepithelial cells (hSMECs) and stem/progenitor cells (hS/PCs) isolated from normal salivary gland tissues. Phenotypic characterization of isolated hSMECs confirmed that a myoepithelial cell phenotype consistent with that from other exocrine tissues was maintained over multiple passages of culture. Additionally, hSMECs secreted basement membrane proteins, expressed adrenergic and cholinergic neurotransmitter receptors, and released intracellular calcium [Ca2+i] in response to parasympathetic agonists. In a collagen I contractility assay, activation of contractile machinery was observed in isolated hSMECs treated with parasympathetic agonists. Recombination of hSMECs with assembled hS/PC spheroids in a microwell system was used to create microtissues resembling secretory complexes of the salivary gland. We conclude that the engineered salivary gland microtissue complexes provide a physiologically relevant model for both mechanistic studies and as a building block for the successful engineering of the salivary gland for restoration of salivary function in patients suffering from hyposalivation.

Primary Salivary Human Stem/Progenitor Cells Undergo Microenvironment-Driven Acinar-Like Differentiation in Hyaluronate Hydrogel Culture.

Radiotherapy for head and neck cancer often has undesirable effects on salivary glands that lead to xerostomia or severe dry mouth, which can increase oral infections. Our goal is to engineer functional, three-dimensional (3D) salivary gland neotissue for autologous implantation to provide permanent relief. An immediate need exists to obtain autologous adult progenitor cells as the use of embryonic and induced pluripotent stem cells potentially pose serious risks such as teratogenicity and immunogenic rejection. Here, we report an expandable population of primary salivary human stem/progenitor cells (hS/PCs) that can be reproducibly and scalably isolated and propagated from tissue biopsies. These cells have increased expression of progenitor markers (K5, K14, MYC, ETV4, ETV5) compared with differentiation markers of the parotid gland (acinar: MIST1/BHLHA15 and AMY1A; ductal: K19 and TFCP2L1). Isolated hS/PCs grown in suspension formed primary and secondary spheres and could be maintained in long-term 3D hydrogel culture. When grown in a customized 3D modular hyaluronate-based hydrogel system modified with bioactive basement membrane-derived peptides, levels of progenitor markers, indices of proliferation, and viability of hS/PCs were enhanced. When appropriate microenvironmental cues were provided in a controlled manner in 3D, such as stimulation with ß-adrenergic and cholinergic agonists, hS/PCs differentiated into an acinar-like lineage, needed for saliva production. We conclude that the stem/progenitor potential of adult hS/PCs isolated without antigenic sorting or clonal expansion in suspension, combined with their ability to differentiate into specialized salivary cell lineages in a human-compatible culture system, makes them ideal for use in 3D bioengineered salivary gland applications. Stem Cells Translational Medicine 2017;6:110-120.

Selective Targeting of Myeloid-Derived Suppressor Cells in Cancer Patients Using DS-8273a, an Agonistic TRAIL-R2 Antibody.

Purpose: Myeloid-derived suppressor cells (MDSC) are one of the major contributors to immune suppression in cancer. We recently have demonstrated in preclinical study that MDSCs are sensitive to TRAIL receptor 2 (TRAIL-R2) agonist. The goal of this study was to clinically test the hypothesis that targeting TRAIL-R2 can selectively eliminate MDSCs.Experimental Design: The TRAIL-R2 agonistic antibody (DS-8273a) has been tested in 16 patients with advanced cancers enrolled in a phase I trial. The antibody (24 mg/kg) was administered intravenously once every 3 weeks till disease progression, unacceptable toxicities, or withdrawal of consent. The safety and the presence of various populations of myeloid and lymphoid cells in peripheral blood and tumor tissues were evaluated.Results: The treatment was well tolerated with only mild to moderate adverse events attributable to the study drug. Treatment with DS-8273a resulted in reduction of the elevated numbers of MDSCs in the peripheral blood of most patients to the levels observed in healthy volunteers. However, in several patients, MDSCs rebounded back to the pretreatment level by day 42. In contrast, DS-8273a did not affect the number of neutrophils, monocytes, and other populations of myeloid and lymphoid cells. Decrease in MDSCs inversely correlated with the length of progression-free survival. In tumors, DS-8273a treatment resulted in a decrease of MDSCs in 50% of the patients who were able to provide pre- and on-treatment biopsies.Conclusions: Targeting TRAIL-R2 resulted in elimination of different populations of MDSCs without affecting mature myeloid or lymphoid cells. These data support the use of this antibody in combination immmunotherapy of cancer. Clin Cancer Res; 23(12); 2942-50. ©2016 AACR.

Warthin tumor within the superficial lobe of the parotid gland: a suggested criterion for diagnosis.

The location of Warthin tumor (WT) in the parotid gland impacts the surgical approach and may be indicative of the elusive origin of this intriguing entity. Location in the deep versus superficial lobe of the gland is not directly addressed when defining WT characteristics. Our observation, of rare occurrence of deep lobe WT, if at all, led to the current investigation. The study design is cohort study. This is a retrospective chart review of all patients undergoing parotidectomy for WT in two tertiary academic referral centers: the Sheba Medical Center (SMC), Israel, and the Christiana Care (CC), Newark, Delaware, USA. 122 consecutive adult patients underwent parotidectomy for WT (72 from SMC and 50 from CC). Seventy percent were males, with a mean age of 60.6 years. Bilateral WT or multi-centric WT were found in 9.8 and 17.2% of the cases, respectively. In one case, the tumor was described as originating in the deep lobe. In all other cases, the tumor originated and was limited to the superficial lobe. 99.2% of WT originated in the superficial lobe, corresponding with the few reports directly addressing its location in the gland. The reason for the tumor to be limited almost uniformly to the superficial lobe is unknown, and could be related to the etiopathogenesis of this elusive entity. We suggest adding tumor location within the superficial lobe to the common characteristics of WT (male, smoking, and lower pole) that serve as "common criterion" while evaluating a parotid lesion.

Tuning Hydrogel Properties to Promote the Assembly of Salivary Gland Spheroids in 3D.

Current treatments for chronic xerostomia, or "dry mouth", do not offer long-term therapeutic benefits for head and neck cancer survivors previously treated with curative radiation. Towards the goal of creating tissue-engineered constructs for the restoration of salivary gland functions, we developed new hyaluronic acid (HA)-based hydrogels using thiolated HA (HA-SH) and acrylated HA (HA-AES) with a significant molecular weight mismatch. Four hydrogel formulations with varying HA concentration, (1-2.4 wt%) and thiol/acrylate ratios (2/1 to 36/1) and elastic moduli (G': 35 to 1897 Pa, 2 h post-mixing) were investigated. In our system, thiol/acrylate reaction was initiated rapidly upon mixing of HA-SH/HA-AES to establish thioether crosslinks with neighboring ester groups, and spontaneous sulfhydryl oxidation occurred slowly over several days to install a secondary network. The concurrent reactions cooperatively create a cell-permissive network to allow for cell expansion and aggregation. Multicellular spheroids formed readily from a robust ductal epithelial cell line (Madin-Darby Canine Kidney, MDCK cells) in all hydrogel formulations investigated. Primary salivary human stem/progenitor cells (hS/PCs), on the other hand, are sensitive to the synthetic extracellular environment, and organized acini-like structures with an average diameter of 50 µm were obtained only in gels with G' ≤ 216 Pa and a thiol/acrylate ratio ≥18. The spheroid size and size distribution were dependent on the HA content in the hydrogel. Cells in hS/PC spheroids formed tight junctions (occludin), remained viable and proliferative, secreted structural proteins (collagen IV and laminin) found in the basement membrane and maintained key stem/progenitor markers. We conclude that incorporation of time-dependent, dynamic features into a covalently crosslinked HA network produces an adaptable hydrogel framework that promotes hS/PC assembly and supports early aspects of salivary morphogenesis, key to reconstitution of a fully functional implantable salivary gland.

Recurrent Benign Salivary Gland Neoplasms.

The most important causes of recurrence of benign pleomorphic adenoma are enucleation with intraoperative spillage and incomplete tumor excision in association with characteristic histologic findings for the lesion (incomplete pseudocapsule and the presence of pseudopodia). Most recurrent pleomorphic adenomas (RPAs) are multinodular. MRI is the imaging method of choice for their assessment. Nerve integrity monitoring may reduce morbidity of RPA surgery. Although treatment of RPA must be individualized, total parotidectomy is generally recommended given the multicentricity of the lesions. However, surgery alone may be inadequate for controlling RPA over the long term. There is growing evidence from retrospective series that postoperative radiotherapy results in significantly better local control. A high percentage of RPAs are incurable. All patients should therefore be informed about the possibility of needing multiple treatment procedures, with possible impairment of facial nerve function, and radiation therapy for RPA. Reappearance of Warthin tumor is a metachronous occurrence of a new focus or residual incomplete excision of all primary multicentric foci of Warthin tumor. Selected cases can be observed. Conservative surgical management can include partial superficial parotidectomy or extracapsular dissection. Not uncommonly, other major and minor salivary gland neoplasms, including myoepithelioma, basal cell adenoma, oncocytoma, canalicular adenoma, cystadenoma, and ductal papilloma, follow an indolent course after surgical resection, with rare cases of recurrence.

Outcome of thyroid gene expression classifier testing in clinical practice.

OBJECTIVES/HYPOTHESIS: Gene expression classifiers can safely reduce diagnostic thyroid surgery for fine-needle aspiration cytology (FNAC) indeterminate thyroid nodules. STUDY DESIGN: Retrospective review, single-institution, single-practice surgeon. METHODS: Three-year retrospective review of indeterminate FNAC that went on to gene expression classifier testing. RESULTS: A total of 520 patients met American Thyroid Association guideline criteria for surgeon-performed ultrasound-guided FNAC for a thyroid nodule with on-site cytopathology. The indeterminate (Bethesda III or IV) FNAC rate was 9%. Prevalence of malignancy in FNAC indeterminate was 21%. Thirty-two cases went on to gene expression classifier testing. Fourteen were benign, 15 suspicious, and three with no result. CONCLUSIONS: Benign gene expression classifier testing had an estimated negative predictive value of 100% during the study period. These patients have been observed for a mean and median duration of 14 and 7 months, respectively. In this small series, 14 of 29 patients with indeterminate FNAC were spared diagnostic surgery. LEVEL OF EVIDENCE: 4.

Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) are important regulators of immune responses in cancer and have been directly implicated in promotion of tumor progression. However, the heterogeneity of these cells and lack of distinct markers hampers the progress in understanding of the biology and clinical importance of these cells. Using partial enrichment of PMN-MDSC with gradient centrifugation we determined that low density PMN-MDSC and high density neutrophils from the same cancer patients had a distinct gene profile. Most prominent changes were observed in the expression of genes associated with endoplasmic reticulum (ER) stress. Surprisingly, low-density lipoprotein (LDL) was one of the most increased regulators and its receptor oxidized LDL receptor 1 OLR1 was one of the most overexpressed genes in PMN-MDSC. Lectin-type oxidized LDL receptor 1 (LOX-1) encoded by OLR1 was practically undetectable in neutrophils in peripheral blood of healthy donors, whereas 5-15% of total neutrophils in cancer patients and 15-50% of neutrophils in tumor tissues were LOX-1+. In contrast to their LOX-1- counterparts, LOX-1+ neutrophils had gene signature, potent immune suppressive activity, up-regulation of ER stress, and other biochemical characteristics of PMN-MDSC. Moreover, induction of ER stress in neutrophils from healthy donors up-regulated LOX-1 expression and converted these cells to suppressive PMN-MDSC. Thus, we identified a specific marker of human PMN-MDSC associated with ER stress and lipid metabolism, which provides new insight to the biology and potential therapeutic targeting of these cells.

Targeted Next Generation Sequencing with ThyroSeq v2.1 for Indeterminate Thyroid Nodules in Clinical Practice.

OBJECTIVE: To determine if patients elect molecular testing over diagnostic surgery or repeat fine needle aspiration for indeterminate thyroid nodules. Can ThyroSeq v2.1 molecular testing reduce diagnostic thyroid surgery and rule out cancer? STUDY DESIGN: Retrospective review Setting: Single institution, single-practice surgeon. SUBJECTS AND METHODS: Fifteen month retrospective review of indeterminate thyroid nodules that went on to ThyroSeq v2.1 testing. RESULTS: 286 patients met American Thyroid Association guideline criteria for surgeon- performed, ultrasound-guided fine needle aspiration for a thyroid nodule with on-site cytopathology. The indeterminate (Bethesda III or IV) fine needle aspiration cytology rate was 9.1 percent. Prevalence of malignancy in indeterminate nodules was 19 percent. 26/26 (100 percent) patients with indeterminate thyroid nodules elected molecular testing. 16 patients had no mutation, 9 had one or more mutations, and I had no result. 16 of 25 (64 percent) patients with no mutation elected not to undergo diagnostic surgery for indeterminate thyroid nodules. CONCLUSIONS: Patients demonstrated a strong preference for molecular testing instead of diagnostic thyroid surgery for indeterminate thyroid nodules. All patients in this series, 25/25 (100 percent) with indeterminate thyroid nodules elected molecular testing instead of repeat biopsy or diagnostic thyroid surgery. 16 of 25 (64 percent) patients tested had no mutation. All 16/16 (100 percent) patients with no mutation on ThyroSeq "rule out" testing elected active surveillance rather than surgery or biopsy, reducing diagnostic surgery. The risk of malignancy among mutation negative patients was not definitively established. There are a number of factors currently that may mute the power of "rule in" testing.