APE-1/Ref-1 Inhibition Blocks Malignant Pleural Mesothelioma Cell Proliferation and Migration: Crosstalk between Oxidative Stress and Epithelial Mesenchymal Transition (EMT) in Driving Carcinogenesis and Metastasis.

Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM pathogenesis has been related both to oxidative stress, evoked by and in response to asbestos fibers exposure, and epithelial mesenchymal transition (EMT), an event induced by oxidative stress itself and related to cancer proliferation and metastasis. Asbestos-related primary oxidative damage is counteracted in the lungs by various redox-sensitive factors, often hyperactivated in some cancers. Among these redox-sensitive factors, Apurinic-apyrimidinic endonuclease 1 (APE-1)/Redox effector factor 1 (Ref-1) has been demonstrated to be overexpressed in MPM and lung cancer, but the molecular mechanism has not yet been fully understood. Moreover, asbestos exposure has been associated with induced EMT events, via some EMT transcription factors, such as Twist, Zeb-1 and Snail-1, in possible crosstalk with oxidative stress and inflammation events. To demonstrate this hypothesis, we inhibited/silenced Ref-1 in MPM cells; as a consequence, both EMT (Twist, Zeb-1 and Snail-1) markers and cellular migration/proliferation were significantly inhibited. Taken as a whole, these results show, for the first time, crosstalk between oxidative stress and EMT in MPM carcinogenesis and invasiveness, thus improving the knowledge to better address a preventive and therapeutic approach against this aggressive cancer.

The Epithelial-to-Mesenchymal Transition (EMT) in the Development and Metastasis of Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumor mainly associated with asbestos exposure and is characterized by a very difficult pharmacological approach. One of the molecular mechanisms associated with cancer onset and invasiveness is the epithelial-to-mesenchymal transition (EMT), an event induced by different types of inducers, such as transforming growth factor ß (TGFß), the main inducer of EMT, and oxidative stress. MPM development and metastasis have been correlated to EMT; On one hand, EMT mediates the effects exerted by asbestos fibers in the mesothelium, particularly via increased oxidative stress and TGFß levels evoked by asbestos exposure, thus promoting a malignant phenotype, and on the other hand, MPM acquires invasiveness via the EMT event, as shown by an upregulation of mesenchymal markers or, although indirectly, some miRNAs or non-coding RNAs, all demonstrated to be involved in cancer onset and metastasis. This review aims to better describe how EMT is involved in driving the development and invasiveness of MPM, in an attempt to open new scenarios that are useful in the identification of predictive markers and to improve the pharmacological approach against this aggressive cancer.

Low-risk papillary thyroid microcarcinoma: Optimal management toward a more conservative approach.

The incidence of papillary thyroid microcarcinoma (microPTC) has dramatically increased in the last decades. Most of these tumors remain small and clinically "silent", only small number progress. Although thyroid surgery used to be the only therapeutic approach, recent guidelines now consider active surveillance for low-risk microPTC. For this reason, more accurate risk stratification of microPTC is needed. The optimal management of low-risk microPTC through accurate risk stratification represents a major clinical issue.

Diagnostic Performance of Neck Ultrasonography in the Preoperative Evaluation for Extrathyroidal Extension of Suspicious Thyroid Nodules.

BACKGROUND: A preoperative neck ultrasound (US) is recommended for all patients with suspected thyroid cancer, to identify features potentially changing surgical extent. The extrathyroidal extension (ETE) is considered an indication for total thyroidectomy, but there is limited consensus on its US definition, and the interobserver reliability is low. This study aimed to evaluate the predictive value of neck US for ETE before surgery and to estimate the diagnostic performance of different US findings, evaluated during real-time examinations. METHODS: Patients referred to surgery between November 1, 2015, and May 31, 2019, for a suspicious thyroid cancer underwent a preoperative neck US, with systematic assessment for ETE. Three definitions were tested: very restrictive (capsular disruption with suspicious images of surrounding tissues invasion), restrictive (including also capsular abutment with evidence of capsular disruption), and nonrestrictive (capsular abutment is sufficient). Histopathology report of ETE involving at least soft tissues was considered positive. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated. RESULTS: The study cohort included 128 patients, with 102 (79.7%) confirmed malignancies, and 44 (43.1%) histological ETE. The nonrestrictive definition had good sensitivity (86.4%) but low specificity (29.8%), with an NPV of 80.6%; the restrictive definition had higher specificity (81%), while the very restrictive had specificity and PPV of 100%. CONCLUSIONS: A more extensive surgical approach should not be based on US suspicion of ETE alone, with the possible exception of gross invasion appearance. The absence of any sign of ETE, on the other hand, has a substantial negative predictive value.

IS IT WORTH SUPPRESSING TSH IN LOW- AND INTERMEDIATE-RISK PAPILLARY THYROID CANCER PATIENTS BEFORE THE FIRST DISEASE ASSESSMENT?

OBJECTIVE: Guidelines recommend thyroid-stimulating hormone (TSH) suppression before the first response to treatment assessment in papillary thyroid cancer (PTC) patients. The aim of this study was to assess the rate of structural disease (SD) in low- and intermediate-risk PTC patients according to TSH levels measured 1 year after primary treatment. METHODS: A consecutive, prospective series of low- and intermediate-risk PTC patients with 3-years follow-up was collected. TSH, thyroglobulin (Tg), antithyroglobulin antibodies (TgAb), and neck ultrasonography (US) 1 and 3 years after primary treatment were analyzed. Recurrence risk and disease status at 1 year were defined according to the American Thyroid Association (ATA) guidelines and as the presence or absence of SD after 3 years. Patients were grouped according to TSH level at 1 year: group 1, TSH <0.1 µUI/mL; group 2, TSH 0.1 to 0.5 µUI/mL; group 3, 0.5 to 2 µUI/mL; and group 4 >2 µUI/mL. RESULTS: This study included 263 patients (70.9% female, median age 47.2 years) of whom the risk of recurrence was low in 170 (65%), intermediate-low in 63 (24%), and intermediate-high in 30 (11%). The response to initial treatment at 1 year was excellent in 149 (57%), biochemical incomplete in 18 (7%), indeterminate in 84 (32%), and structural incomplete in 12 (4%). Group 1 consisted of 53 (20%) patients, group 2 of 85 (32%), group 3 of 61 (23%), and group 4 of 64 (24%). The rate of SD at 1 and 3 years from primary treatment was not significantly different between TSH groups. CONCLUSION: TSH suppression before the first response to treatment assessment does not appear to influence the rate of SD evaluated 1 and 3 years after primary treatment. ABBREVIATIONS: ATA = American Thyroid Association; DTC = differentiated thyroid cancer; FTC = follicular thyroid cancer; LT4 = levothyroxine; PTC = papillary thyroid cancer; SD = structural disease; Tg = thyroglobulin; TgAb = antithyroglobulin antibodies; TSH = thyroid-stimulating hormone; US = ultrasonography.

Prognostic role of the CDNK1B V109G polymorphism in multiple endocrine neoplasia type 1.

CDKN1B encodes the cyclin-dependent kinase inhibitor p27/Kip1. CDKN1B mutations and polymorphisms are involved in tumorigenesis; specifically, the V109G single nucleotide polymorphism has been linked to different tumours with controversial results. Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominant syndrome, characterized by the development of different types of neuroendocrine tumours and increased incidence of other malignancies. A clear genotype-phenotype correlation in MEN1 has not been established yet. In this study, we assessed whether the CDKN1B V109G polymorphism was associated with the development of aggressive tumours in 55 consecutive patients affected by MEN1. The polymorphism was investigated by PCR amplification of germline DNA followed by direct sequencing. Baseline and follow-up data of tumour types and their severity were collected and associated with the genetic data. MEN1-related aggressive and other malignant tumours of any origin were detected in 16.1% of wild-type and 33.3% of polymorphism allele-bearing patients (P = NS). The time interval between birth and the first aggressive tumour was significantly shorter in patients with the CDKN1B V109G polymorphism (median 46 years) than in those without (median not reached; P = 0.03). Similarly, shorter was the time interval between MEN1 diagnosis and age of the first aggressive tumour (P = 0.02). Overall survival could not be estimated as 96% patients were still alive at the time of the study. In conclusion, CDKN1B V109G polymorphism seems to play a role in the development of aggressive tumours in MEN1.

Unraveling Novel Strategies in Mesothelioma Treatments Using a Newly Synthetized Platinum(IV) Compound.

Malignant mesothelioma is a rare tumor associated with asbestos exposure. Mesothelioma carcinogenesis is related to enhanced reactive oxygen species (ROS) production and iron overload. Despite the recent advances in biomedical sciences, to date the only available treatments include surgery in a small fraction of patients and platinum-based chemotherapy in combination with pemetrexed. In this view, the purpose of this study was to evaluate the therapeutic potential of the newly synthetized platinum prodrug Pt(IV)Ac-POA compared to cisplatin (CDDP) on human biphasic mesothelioma cell line MSTO-211H using different complementary techniques, such as flow-cytometry, transmission electron microscopy (TEM), and immunocytochemistry. Healthy mesothelial cell lines Met-5A were also employed to assess the cytotoxicity of the above-mentioned compounds. Our in vitro results showed that Pt(IV)Ac-POA significantly interfere with iron metabolisms and more importantly is able to trigger cell death, through different pathways, including ferroptosis, necroptosis, and apoptosis, in neoplastic cells. On the other hand, CDDP triggers mainly apoptotic and necrotic cell death. In conclusion, Pt(IV)Ac-POA may represent a new promising pharmacological agent in the treatment of malignant mesothelioma.

Sorafenib in advanced iodine-refractory differentiated thyroid cancer: efficacy, safety and exploratory analysis of role of serum thyroglobulin and FDG-PET.

CONTEXT: Radioactive iodine is a crucial tool for treatment of differentiated thyroid cancer (DTC). In 5% of cases, DTCs lose I-131 avidity and assume an aggressive behaviour. Treatment options for iodine-refractory DTC are limited. We report the experience of off-label use of the tyrosine kinase inhibitor sorafenib for treatment of advanced iodine-refractory DTC. DESIGN: Patients with progressive DTC refractory to radioactive iodine were treated with sorafenib used off-label independently from their performance status. Primary study end-points were radiological response, progression-free survival (PFS) and safety. Secondary end-points were site-specific radiological response and overall survival (OS). An exploratory analysis of the role of serum thyroglobulin (Tg) and fluorodeoxyglucose (FDG) positron emission tomography (PET) was performed. RESULTS: A total of 17 patients were included in the study. Median follow-up was 15·5 months. Clinical benefit was obtained in 71% of subjects (30% partial response and 41% stable disease). Sorafenib was mostly well tolerated, but a high incidence of fatal events was reported (three patients died from severe bleeding events and two from cardiac arrest). Median PFS was 9 months. Median OS was 10 months. The best responses were observed in lymph nodes and lung. Baseline Tg levels and the Tg response to treatment were correlated to both radiological response and PFS. Baseline FDG-PET assessment and early FDG-PET response were correlated to radiological response. CONCLUSIONS: Sorafenib allows morphological disease control in the majority of patients with iodine-refractory DTC. Progression-free survival and overall survival were lower than in previous studies as a consequence of the worse clinical condition of our patients. Sorafenib is mostly well tolerated but could have been responsible for the reported fatal events. Baseline Tg and the Tg response to treatment could be useful for predicting morphological response and clinical outcome. Early FDG-PET response could be helpful for the timely identification of nonresponding patients.

TGF-ß as Predictive Marker and Pharmacological Target in Lung Cancer Approach.

Lung cancer (LC) represents the leading cause of cancer incidence and mortality worldwide. LC onset is strongly related to genetic mutations and environmental interactions, such as tobacco smoking, or pathological conditions, such as chronic inflammation. Despite advancement in knowledge of the molecular mechanisms involved in LC, this tumor is still characterized by an unfavorable prognosis, and the current therapeutic options are unsatisfactory. TGF-ß is a cytokine that regulates different biological processes, particularly at the pulmonary level, and its alteration has been demonstrated to be associated with LC progression. Moreover, TGF-ß is involved in promoting invasiveness and metastasis, via epithelial to mesenchymal transition (EMT) induction, where TGF-ß is the major driver. Thus, a TGF-ß-EMT signature may be considered a potential predictive marker in LC prognosis, and TGF-ß-EMT inhibition has been demonstrated to prevent metastasis in various animal models. Concerning a LC therapeutic approach, some TGF-ß and TGF-ß-EMT inhibitors could be used in combination with chemo- and immunotherapy without major side effects, thereby improving cancer therapy. Overall, targeting TGF-ß may be a valid possibility to fight LC, both in improving LC prognosis and cancer therapy, via a novel approach that could open up new effective strategies against this aggressive cancer.

Limitations of Chromogranin A in clinical practice.

CONTEXT: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. METHODS: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. RESULTS: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. CONCLUSION: Serum CgA is not helpful for the first-line diagnosis of NEN.

Transforming Growth Factor-ß and Oxidative Stress in Cancer: A Crosstalk in Driving Tumor Transformation.

Cancer metabolism involves different changes at a cellular level, and altered metabolic pathways have been demonstrated to be heavily involved in tumorigenesis and invasiveness. A crucial role for oxidative stress in cancer initiation and progression has been demonstrated; redox imbalance, due to aberrant reactive oxygen species (ROS) production or deregulated efficacy of antioxidant systems (superoxide dismutase, catalase, GSH), contributes to tumor initiation and progression of several types of cancer. ROS may modulate cancer cell metabolism by acting as secondary messengers in the signaling pathways (NF-kB, HIF-1α) involved in cellular proliferation and metastasis. It is known that ROS mediate many of the effects of transforming growth factor ß (TGF-ß), a key cytokine central in tumorigenesis and cancer progression, which in turn can modulate ROS production and the related antioxidant system activity. Thus, ROS synergize with TGF-ß in cancer cell metabolism by increasing the redox imbalance in cancer cells and by inducing the epithelial mesenchymal transition (EMT), a crucial event associated with tumor invasiveness and metastases. Taken as a whole, this review is addressed to better understanding this crosstalk between TGF-ß and oxidative stress in cancer cell metabolism, in the attempt to improve the pharmacological and therapeutic approach against cancer.

Selective Use of Radioactive Iodine Therapy for Papillary Thyroid Cancers With Low or Lower-Intermediate Recurrence Risk.

CONTEXT: Current guidelines recommend a selective use of radioiodine treatment (RAI) for papillary thyroid cancer (PTC). OBJECTIVE: This work aimed to determine how policy changes affect the use of RAI and the short-term outcomes of patients. METHODS: A retrospective analysis of longitudinal data was conducted in an academic referral center of patients with nonaggressive PTC variants; no extrathyroidal invasion or limited to soft tissues, no distant metastases, and 5 or fewer central-compartment cervical lymph node metastases. In cohort 1, standard treatments were total thyroidectomy and RAI (May 2005-June 2011); in cohort 2 decisions on RAI were deferred for approximately 12 months after surgery (July 2011-December 2018). Propensity score matching was used to adjust for sex, age, tumor size, lymph node status, and extrathyroidal extension. Intervention included immediate RAI or deferred choice. Main outcome measures were responses to initial treatment during 3 or more years of follow-up. RESULTS: In cohort 1, RAI was performed in 50 of 116 patients (51.7%), whereas in cohort 2, it was far less frequent: immediately in 10 of 156 (6.4%), and in 3 more patients after the first follow-up data. The frequencies of structural incomplete response were low (1%-3%), and there were no differences between the 2 cohorts at any follow-up visit. Cohort 2 patients had higher rates of "gray-zone responses" (biochemical incomplete or indeterminate response). CONCLUSION: Selective use of RAI increases the rate of patients with "uncertain" status during early follow-up. The rate of structural incomplete responses remains low regardless of whether RAI is used immediately. Patients should be made aware of the advantages and drawbacks of omitting RAI.

Contemporary Thyroid Nodule Evaluation and Management.

CONTEXT: Approximately 60% of adults harbor 1 or more thyroid nodules. The possibility of cancer is the overriding concern, but only about 5% prove to be malignant. The widespread use of diagnostic imaging and improved access to health care favor the discovery of small, subclinical nodules and small papillary cancers. Overdiagnosis and overtreatment is associated with potentially excessive costs and nonnegligible morbidity for patients. EVIDENCE ACQUISITION: We conducted a PubMed search for the recent English-language articles dealing with thyroid nodule management. EVIDENCE SYNTHESIS: The initial assessment includes an evaluation of clinical risk factors and sonographic examination of the neck. Sonographic risk-stratification systems (e.g., Thyroid Imaging Reporting and Data Systems) can be used to estimate the risk of malignancy and the need for biopsy based on nodule features and size. When cytology findings are indeterminate, molecular analysis of the aspirate may obviate the need for diagnostic surgery. Many nodules will not require biopsy. These nodules and those that are cytologically benign can be managed with long-term follow-up alone. If malignancy is suspected, options include surgery (increasingly less extensive), active surveillance or, in selected cases, minimally invasive techniques. CONCLUSION: Thyroid nodule evaluation is no longer a 1-size-fits-all proposition. For most nodules, the likelihood of malignancy can be confidently estimated without resorting to cytology or molecular testing, and low-frequency surveillance is sufficient for most patients. When there are multiple options for diagnosis and/or treatment, they should be discussed with patients as frankly as possible to identify an approach that best meets their needs.

Fournier's gangrene during lenvatinib treatment: A case report.

Fournier's gangrene is a rare and severe complication reported in patients with cancer treated with antiangiogenic drugs, most frequently with bevacizumab. The present report describes the case of an 80-year-old man with radioactive iodine-refractory metastatic thyroid cancer treated with lenvatinib (an oral multikinase inhibitor with antiangiogenic properties) who developed Fournier's gangrene in the absence of other known risk factors. To the best of our knowledge, this is the first case described during treatment with lenvatinib. The condition was likely due to a perturbation of vascular endothelial cells of the skin due to the inhibition of VEGF/VEGFR signaling. Fournier's gangrene may be a class effect of antiangiogenic treatment that clinicians should be aware of, as early diagnosis and treatment are associated with an improved outcome.

Cancer Care During COVID-19 Era: The Quality of Life of Patients With Thyroid Malignancies.

Background: The Covid-19 pandemic's potential psychological impact has been widely discussed on the basis of expert opinion and previous experience with emergencies of this type. We conducted a survey of cancer patients to explore more objectively the outbreak's impact on their emotional well-being and quality of life. Methods: Between March 18 and April 4, 2020, at an endocrine cancer center in Rome, Italy, 137 patients were asked to complete an online 6-item questionnaire developed by our staff to explore the emotional effects of the Covid-19 outbreak in Italy (Covid-19 Emotional Impact Survey, C-19EIS). For validation purposes, we also asked participants to complete an online version of the validated Italian translation of the EORTC QLQ-C30 questionnaire. Responses were analyzed in relation to responders' age, sex, and clinical status (advanced/metastatic disease undergoing systemic treatment vs. stable metastatic thyroid cancer in active surveillance vs. low-risk thyroid cancers with no evidence of structural disease during standard follow-up). Results: Response rates were high (51% for the C-19EIS, 44.5% for the EORTC QLQ-C30). Overall C-19EIS scores indicated high concern over the outbreak (median 8/12). Scores were higher in women (8 [IQR 5-9] vs. 6 [IQR 5-8] in men; p = 0.048) and in patients <65 years (8 [IQR 5-9] vs. 6 [IQR 4-8] in older patients; p = 0.013). No differences emerged across clinical status groups. C-19EIS scores were inversely correlated with the EORTC QLQ-C30 Emotional function subscale (rho -0.69; p < 0.001). Conclusions: There is objective evidence that the Covid-19 outbreak is causing substantial emotional distress among cancer patients, regardless of their disease severity or current health-care needs.

Sonographic Risk Stratification Systems for Thyroid Nodules as Rule-Out Tests in Older Adults.

Ultrasonographic risk-stratification systems (RSS), including various Thyroid Imaging Reporting and Data Systems (TIRADS), were proposed to improve reporting and reduce the number of fine-needle aspiration biopsies. However, age might be a confounder since some suspicious ultrasonographic features lack specificity in elderly patients. We aimed to investigate whether the diagnostic performance of the RSS varied between age groups. All patients consecutively referred for thyroid biopsy between November 1, 2015, and March 10, 2020, were included. The malignancy risk of each nodule was estimated according to five RSS: the American Association of Clinical Endocrinologists/American College of Endocrinology/Associazione Medici Endocrinologi guidelines, the American College of Radiology (ACR) TIRADS, the American Thyroid Association guidelines, the European TIRADS, and the Korean TIRADS. Overall, 818 nodules (57 malignant) were evaluated. The malignancy rate was higher in patients ≤ 65 years (8.1%) than in patients > 65 years (3.8%; p = 0.02). All RSS confirmed a significant discriminative performance in both age groups, with a negative predictive value of 100% in patients > 65 years, although specificity was lower in older patients. The ACR TIRADS was the best performing in both age groups. RSS could avoid a sizable number of biopsies when applied as rule-out tests in elderly patients.

Endocrine surgery during COVID-19 pandemic: do we need an update of indications in Italy?

The ongoing spread of the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a significant threat to global health. As the coronavirus outbreak began spreading, hospitals were forced to relocate resources to treat the growing number of COVID-19 patients. As a consequence, doctors across the country canceled tens of thousands of nonurgent surgeries. However, recognizing that the COVID-19 situation may be highly variable and fluid in different communities across the country, elective surgery could be still allowed in some centers for patients included in the high-priority class. The majority of endocrine disorders requiring surgical treatment in patients identifiable as first-priority class, or needing hospitalization within 30 days, are generally represented by malignant thyroid tumors, hyperthyroidism, hyperparathyroidism, and some adrenal disorders. The need for urgent intervention is evaluated on a case-by-case basis according to the severity of the symptoms, the likelihood of progression, and global clinical judgment. On the basis of the above indications, during the last 4 weeks, we performed 18 planned surgical treatments in patients with thyroid cancer (total thyroidectomies, plus lymph node dissection if needed) or multinodular toxic goiter. In no case, postoperative ventilatory support was needed, and the average hospital stay was 3 days. The negative COVID-19 status for all the treated patients was appropriately evaluated beforehand. Nobody knows how long the current COVID-19 pandemic will be lasting. Certainly, we will be requested in the next future to incrementally offer surgical services for endocrine disorders that have been deferred for the COVID-19 pandemic.

Correction to: Exploring the molecular insights of concurrent composite mucoepidermoid carcinoma and papillary thyroid carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue.

PURPOSE: The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. METHODS: We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. RESULTS: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2-5% mutant allele frequency. CONCLUSIONS: This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Loss of Function SETD2 Mutations in Poorly Differentiated Metastases from Two Hürthle Cell Carcinomas of the Thyroid.

Hürthle cell carcinomas (HCC) are rare differentiated thyroid cancers that display low avidity for radioactive iodine and respond poorly to kinase inhibitors. Here, using next-generation sequencing, we analyzed the mutational status of primary tissue and poorly differentiated metastatic tissue from two HCC patients. In both cases, metastatic tissues harbored a mutation of SETD2, each resulting in loss of the SRI and WW domains of SETD2, a methyltransferase that trimethylates H3K36 (H3K36me3) and also interacts with p53 to promote its stability. Functional studies of the novel p.D1890fs6* mutation (case 1) revealed significantly reduced H3K36me3 levels in SETD2-mutated tissue and primary cell cultures and decreased levels of the active form of p53. Restoration of SETD2-wildtype expression in the SETD2-mutant cells significantly reduced the expression of four well-known stemness markers (OCT-4, SOX2, IPF1, Goosecoid). These findings suggest potential roles for SETD2 loss-of-function mutations in HCC progression, possibly involving p53 destabilization and promotion of stemness. Their prevalence and potential treatment implications in thyroid cancer, especially HCC, require further study.