Defining the In Vivo Role of mTORC1 in Thyrocytes by Studying the TSC2 Conditional Knockout Mouse Model.

Background: The thyroid gland is susceptible to abnormal epithelial cell growth, often resulting in thyroid dysfunction. The serine-threonine protein kinase mechanistic target of rapamycin (mTOR) regulates cellular metabolism, proliferation, and growth through two different protein complexes, mTORC1 and mTORC2. The PI3K-Akt-mTORC1 pathway's overactivity is well associated with heightened aggressiveness in thyroid cancer, but recent studies indicate the involvement of mTORC2 as well. Methods: To elucidate mTORC1's role in thyrocytes, we developed a novel mouse model with mTORC1 gain of function in thyrocytes by deleting tuberous sclerosis complex 2 (TSC2), an intracellular inhibitor of mTORC1. Results: The resulting TPO-TSC2KO mice exhibited a 70-80% reduction in TSC2 levels, leading to a sixfold increase in mTORC1 activity. Thyroid glands of both male and female TPO-TSC2KO mice displayed rapid enlargement and continued growth throughout life, with larger follicles and increased colloid and epithelium areas. We observed elevated thyrocyte proliferation as indicated by Ki67 staining and elevated cyclin D3 expression in the TPO-TSC2KO mice. mTORC1 activation resulted in a progressive downregulation of key genes involved in thyroid hormone biosynthesis, including thyroglobulin (Tg), thyroid peroxidase (Tpo), and sodium-iodide symporter (Nis), while Tff1, Pax8, and Mct8 mRNA levels remained unaffected. NIS protein expression was also diminished in TPO-TSC2KO mice. Treatment with the mTORC1 inhibitor rapamycin prevented thyroid mass expansion and restored the gene expression alterations in TPO-TSC2KO mice. Although total thyroxine (T4), total triiodothyronine (T3), and TSH plasma levels were normal at 2 months of age, a slight decrease in T4 and an increase in TSH levels were observed at 6 and 12 months of age while T3 remained similar in TPO-TSC2KO compared with littermate control mice. Conclusions: Our thyrocyte-specific mouse model reveals that mTORC1 activation inhibits thyroid hormone (TH) biosynthesis, suppresses thyrocyte gene expression, and promotes growth and proliferation.

DICER1 RNase IIIb domain mutations trigger widespread miRNA dysregulation and MAPK activation in pediatric thyroid cancer.

DICER1 is a highly conserved RNase III endoribonuclease essential for the biogenesis of single-stranded mature microRNAs (miRNAs) from stem-loop precursor miRNAs. Somatic mutations in the RNase IIIb domain of DICER1 impair its ability to generate mature 5p miRNAs and are believed to drive tumorigenesis in DICER1 syndrome-associated and sporadic thyroid tumors. However, the DICER1-driven specific changes in miRNAs and resulting changes in gene expression are poorly understood in thyroid tissue. In this study, we profiled the miRNA (n=2,083) and mRNA (n=2,559) transcriptomes of 20 non-neoplastic, 8 adenomatous and 60 pediatric thyroid cancers (13 follicular thyroid cancers [FTC] and 47 papillary thyroid cancers [PTC]) of which 8 had DICER1 RNase IIIb mutations. All DICER1-mutant differentiated thyroid cancers (DTC) were follicular patterned (six follicular variant PTC and two FTC), none had lymph node metastasis. We demonstrate that DICER1 pathogenic somatic mutations were associated with a global reduction of 5p-derived miRNAs, including those particularly abundant in the non-neoplastic thyroid tissue such as let-7 and mir-30 families, known for their tumor suppressor function. There was also an unexpected increase of 3p miRNAs, possibly associated with DICER1 mRNA expression increase in tumors harboring RNase IIIb mutations. These abnormally expressed 3p miRNAs, which are otherwise low or absent in DICER1-wt DTC and non-neoplastic thyroid tissues, make up exceptional markers for malignant thyroid tumors harboring DICER1 RNase IIIb mutations. The extensive disarray in the miRNA transcriptome results in gene expression changes, which were indicative of positive regulation of cell-cycle. Moreover, differentially expressed genes point to increased MAPK signaling output and loss of thyroid differentiation comparable to the RAS-like subgroup of PTC (as coined by The Cancer Genome Atlas), which is reflective of the more indolent clinical behavior of these tumors.

Development of Novel Murine BRAFV600E-Driven Papillary Thyroid Cancer Cell Lines for Modeling of Disease Progression and Preclinical Evaluation of Therapeutics.

The Cancer Genome Atlas study in thyroid cancer exposed the genomic landscape of ~500 PTCs and revealed BRAFV600E-mutant tumors as having different prognosis, contrasting indolent cases and those with more invasive disease. Here, we describe the generation and characterization of six novel BRAFV600E-driven papillary thyroid cancer (PTC) cell lines established from a BrafV600E+/-/Pten+/-/TPO-Cre mouse model that spontaneously develop thyroid tumors. The novel cell lines were obtained from animals representing a range of developmental stages and both sexes, with the goal of establishing a heterogeneous panel of PTC cell lines sharing a common driver mutation. These cell lines recapitulate the genetics and diverse histopathological features of BRAFV600E-driven PTC, exhibiting differing degrees of growth, differentiation, and invasive potential that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We demonstrate that these cell lines can be used for a variety of in vitro applications and can maintain the potential for in vivo transplantation into immunocompetent hosts. We believe that these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and will lead to the development of more personalized diagnostic and treatment strategies for BRAFV600E-driven PTC.

Platelet findings in 22q11.2 deletion syndrome correlate with disease manifestations but do not correlate with GPIb surface expression.

Prior studies have demonstrated that patients with chromosome 22q11.2 deletion syndrome (22q11.2DS) have lower platelet counts (PC) compared to non-deleted populations. They also have an increased mean platelet volume. The mechanism for this has been postulated to be haploinsufficiency of the GPIBB gene. We examined platelet parameters, deletion size and factors known to influence counts, including status of thyroid hormone and congenital heart disease (CHD), in a population of 825 patients with 22q11.2DS. We also measured surface expression of GPIB-IX complex by flow cytometry. The major determinant of PC was deletion status of GP1BB, regardless of surface expression or other factors. Patients with nested distal chromosome 22q11.2 deletions (those with GP1BB present) had higher PCs than those with proximal deletions where GP1BB is deleted. Patients with 22q11.2DS also demonstrated an accelerated PC decrease with age, occurring in childhood. These data demonstrate that genes within the proximal deletion segment drive PC differences in 22q11.2DS and suggest that PC reference ranges may need to be adjusted for age and deletion size in 22q11.2DS populations. Bleeding did not correlate with either platelet count or GPIb expression. Further studies into drivers of expression of GPIb and associations with severe thrombocytopenia and immune thrombocytopenia are needed to inform clinical care.

Survivorship, Quality of Life, and Transition to Adult Care for Pediatric and Adolescent Thyroid Cancer Survivors.

The importance of long-term survivorship care to reduce survivor and family anxiety and burden, decrease emergency visits and health care costs, improve knowledge, as well as implement timely surveillance is widely accepted. Most childhood cancer survivors suffer from an increased number of medical and psychosocial comorbidities as they age and are at a higher risk for early mortality, which differs by cancer diagnosis. Childhood thyroid cancer survivors fall within this spectrum. Some have significant complications and/or late effects from treatment, whereas others have no long-term medical late effects, but almost all will require life-long thyroid hormone replacement therapy. Therefore, providing survivorship and transitional care, including a survivorship and/or transitional care plan (SCP/TCP), as well as periodically assessing the needs and quality of life for the patient and their family, should be implemented for our young thyroid cancer survivors.

Clinicopathologic Characteristics of Pediatric Follicular Variant of Papillary Thyroid Carcinoma Subtypes: A Retrospective Cohort Study.

Introduction: Follicular patterned thyroid nodules with nuclear features of papillary thyroid carcinoma (PTC) encompass a range of diagnostic categories with varying risks of metastatic behavior. Subtypes include the invasive encapsulated follicular variant of PTC (Ienc-fvPTC) and infiltrative fvPTC (inf-fvPTC), with tumors lacking invasive features classified as noninvasive follicular thyroid neoplasms with papillary-like features (NIFTPs). This study aimed to report the clinical and histological features of pediatric cases meeting criteria for these histological subtypes, with specific focus on Ienc-fvPTC and inf-fvPTC. Methods: In this retrospective cohort study, pediatric patients with thyroid neoplasms showing follicular patterned growth and nuclear features of PTC noted on surgical pathology between January 2010 and January 2021 were retrospectively reviewed and classified according to the recent 2022 World Health Organization (WHO) criteria. Clinical and histopathologic parameters were described for NIFTP, Ienc-fvPTC, and inf-fvPTC subtypes, with specific comparison of Ienc-fvPTC and inf-fvPTC cases. Results: The case cohort included 42 pediatric patients, with 6 (14%), 25 (60%), and 11 (26%) patients meeting criteria for NIFTP, Ienc-fvPTC, and inf-fvPTC, respectively. All cases were rereviewed, and 5 patients originally diagnosed with Ienc-fvPTC before 2017 were reappraised as having NIFTPs. The NIFTP cases were encapsulated tumors without invasive features, lymph node or distant metastasis, or disease recurrence. Ienc-fvPTC tumors demonstrated clearly demarcated tumor capsules and capsular/vascular invasion, while inf-fvPTC tumors displayed infiltrative growth lacking a capsule. inf-fvPTC cases had increased prevalence of malignant preoperative cytology, lymph node metastasis, and distant metastasis (p < 0.01). These cases were treated with total thyroidectomy, lymph node dissection, and subsequent radioactive iodine therapy. Preliminary genetic findings suggest a predominance of fusions in inf-fvPTC cases versus point mutations in Ienc-fvPTC (p = 0.02). Conclusions: Pediatric NIFTP and fvPTC subtypes appear to demonstrate alignment between clinical and histological risk stratification, with indolent behavior in Ienc-fvPTC and invasive features in inf-fvPTC tumors.

Indeterminate Thyroid Fine-Needle Aspirations in Pediatrics: Exploring Clinicopathologic Features and Utility of Molecular Profiling.

INTRODUCTION: The diagnostic utility of molecular profiling for the evaluation of indeterminate pediatric thyroid nodules is unclear. We aimed to assess pediatric cases with indeterminate thyroid fine-needle aspiration (FNA) alongside clinicopathologic features and mutational analysis. METHODS: A retrospective review of 126 patients with indeterminate cytology who underwent FNA between January 2010 and December 2021 at the Children's Hospital of Philadelphia was performed. Indeterminate cases defined by The Bethesda System for Reporting Thyroid Cytopathology (AUS/FLUS or TBSRTC III; FN/SFN or TBSRTC IV; SM or TBSRTC V) were correlated to clinicopathologic and genetic characteristics. RESULTS: Of the 114 surgical cases, 48% were malignant, with the majority of malignant cases diagnosed as follicular variant of papillary thyroid carcinoma (28/55). Risk of malignancy increased with TBSRTC category: 23% for AUS/FLUS, 51% for FN/SFN, and 100% for SM nodules. There were significant differences in surgical approach (p < 0.01), performance of lymph node dissection (p < 0.01), histological diagnosis (p < 0.01), primary tumor focality/laterality (p = 0.04), and lymphatic invasion (p = 0.02) based on TBSRTC classification, with resultant differences in post-surgical risk stratification per American Thyroid Association (ATA) Pediatric Guidelines (p = 0.01). Approximately 89% (49/55) of cases were classified as ATA low risk, and 5 of 6 patients with ATA intermediate- or high-risk disease had SM cytology. Somatic molecular testing was performed in 40% (51/126) of tumors; 77% (27/35) of malignant cases and 38% (6/16) of benign cases harbored driver alteration(s). Of the driver-positive malignant cases, 52% (14/27) were associated with low risk (DICER1, PTEN, RAS, and TSHR mutations), 33% (9/27) were associated with high risk (BRAF mutations and ALK, NTRK, and RET fusions), and 15% (4/27) had unreported risk for invasive disease (APC, BLM, and PPM1D mutations and TG-FGFR1 fusion). Incidence of high-risk drivers increased with TBSRTC category. Approximately 23% (8/35) of patients harboring thyroid malignancy did not have an identifiable driver alteration. CONCLUSIONS: Molecular analysis is useful to discriminate benign and malignant thyroid nodules with indeterminate cytology. Patients with driver genetic alteration(s) and indeterminate cytology should consider surgical management secondary to the high incidence (82%; 27/33) of thyroid malignancy in these patients.

Hormonal Crosstalk Between Thyroid and Breast Cancer.

Differentiated thyroid cancer and breast cancer account for a significant portion of endocrine-related malignancies and predominately affect women. As hormonally responsive tissues, the breast and thyroid share endocrine signaling. Breast cells are responsive to thyroid hormone signaling and are affected by altered thyroid hormone levels. Thyroid cells are responsive to sex hormones, particularly estrogen, and undergo protumorigenic processes upon estrogen stimulation. Thyroid and sex hormones also display significant transcriptional crosstalk that influences oncogenesis and treatment sensitivity. Obesity-related adipocyte alterations-adipocyte estrogen production, inflammation, feeding hormone dysregulation, and metabolic syndromes-promote hormonal alterations in breast and thyroid tissues. Environmental toxicants disrupt endocrine systems, including breast and thyroid homeostasis, and influence pathologic processes in both organs through hormone mimetic action. In this brief review, we discuss the hormonal connections between the breast and thyroid and perspectives on hormonal therapies for breast and thyroid cancer. Future research efforts should acknowledge and further explore the hormonal crosstalk of these tissues in an effort to further understand the prevalence of thyroid and breast cancer in women and to identify potential therapeutic options.

Surgical outcomes in survivors of childhood cancer undergoing thyroidectomy: A single-institution experience.

BACKGROUND: Childhood cancer survivors (CCS) are at increased risk for thyroid disease, and many require definitive management with thyroid surgery. Despite this, there is limited evidence on surgical outcomes among CCS. We sought to evaluate postoperative outcomes at our institution among CCS undergoing thyroid surgery compared to patients without a history of primary childhood malignancy. PROCEDURE: Medical records were reviewed for 638 patients treated at the Children's Hospital of Philadelphia Pediatric Thyroid Center between 2009 and 2020. Rates of surgical complications, including recurrent laryngeal nerve (RLN) paralysis and hypoparathyroidism, among CCS were compared to patients with sporadic/familial thyroid cancer, Graves' disease, and other benign thyroid conditions. Operative time and intraoperative parathyroid hormone levels were also evaluated. RESULTS: There were no significant differences in long-term surgical complication rates, such as permanent RLN paralysis and hypoparathyroidism, between CCS and patients without a history of primary childhood malignancy (all p > .05). For all surgical outcomes, there were no significant differences in complication rates when CCS were compared to those undergoing surgery for sporadic/familial thyroid cancer or Graves' disease (all p > .05). CCS with benign final pathology had significantly higher rates of transient hypoparathyroidism compared to patients with benign thyroid conditions (p < .001). CONCLUSIONS: Our study suggests that CCS are not at higher risk of long-term complications from thyroid surgery when treated by high-volume surgeons within a multidisciplinary team.

NTRK-fusions in pediatric thyroid tumors: Current state and future perspectives.

Pediatric and adult papillary thyroid cancer (PTC) share many similar oncogenic drivers, but differ in the pathological features and outcomes of the disease. The most frequent genetic alterations in adult PTCs are mutually exclusive point mutations in BRAF or the RAS family. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are the most common genetic alterations observed. This review of the literature describes the current state of translational research in pediatric NTRK-driven thyroid cancer and highlights opportunities to improve our understanding and current models of pediatric PTC.

Fusion Oncogenes Are Associated With Increased Metastatic Capacity and Persistent Disease in Pediatric Thyroid Cancers.

PURPOSE: In 2014, data from a comprehensive multiplatform analysis of 496 adult papillary thyroid cancer samples reported by The Cancer Genome Atlas project suggested that reclassification of thyroid cancer into molecular subtypes, RAS-like and BRAF-like, better reflects clinical behavior than sole reliance on pathologic classification. The aim of this study was to categorize the common oncogenic variants in pediatric differentiated thyroid cancer (DTC) and investigate whether mutation subtype classification correlated with the risk of metastasis and response to initial therapy in pediatric DTC. METHODS: Somatic cancer gene panel analysis was completed on DTC from 131 pediatric patients. DTC were categorized into RAS-mutant (H-K-NRAS), BRAF-mutant (BRAF p.V600E), and RET/NTRK fusion (RET, NTRK1, and NTRK3 fusions) to determine differences between subtype classification in regard to pathologic data (American Joint Committee on Cancer TNM) as well as response to therapy 1 year after initial treatment had been completed. RESULTS: Mutation-based subtype categories were significant in most variables, including age at diagnosis, metastatic behavior, and the likelihood of remission at 1 year. Patients with RET/NTRK fusions were significantly more likely to have advanced lymph node and distant metastasis and less likely to achieve remission at 1 year than patients within RAS- or BRAF-mut subgroups. CONCLUSION: Our data support that genetic subtyping of pediatric DTC more accurately reflects clinical behavior than sole reliance on pathologic classification with patients with RET/NTRK fusions having worse outcomes than those with BRAF-mutant disease. Future trials should consider inclusion of molecular subtype into risk stratification.

The clinical aspect of NTRK-fusions in pediatric papillary thyroid cancer.

Although adult and pediatric papillary thyroid cancer (PTC) share similar oncogenic drivers, they differ in the pathological features and outcomes of the disease. In adults with PTC, the most frequent genetic alterations are mutually exclusive point mutations in BRAFV600E or the RAS family with BRAFV600E commonly associated with invasive disease and decreased response to radioiodine therapy. In pediatric PTC, fusion oncogenes involving chromosomal translocations in tyrosine kinase (TK) receptors, most commonly RET and NTRK, are often found in patients with lateral neck and distant metastases. This brief report reviews clinical data from a single-institute's cohort of NTRK-driven pediatric PTC cases with an updated review of the literature and comparison to adult NTRK-driven PTC.

Clinical Course of Early Postoperative Hypothyroidism Following Thyroid Lobectomy in Pediatrics.

Introduction: Thyroid lobectomy reduces risks of surgical complications and need for levothyroxine (LT4). We aimed at identifying the clinical course and risk factors for postlobectomy hypothyroidism to optimize surgical counseling and management in pediatric patients undergoing lobectomy. Methods: Clinical and biochemical presentations pre- and postlobectomy were retrospectively reviewed for 110 patients who underwent thyroid lobectomy between 2008 and 2020 at the Children's Hospital of Philadelphia. Results: Approximately 28.2% of patients (31/110) developed postlobectomy hypothyroidism defined by an elevated thyrotropin (TSH) level, including 24.5% (27/110) with subclinical hypothyroidism (TSH >4.5 and <10.0 mIU/L) and 3.6% (4/110) with overt hypothyroidism (TSH >10.0 mIU/L). LT4 was initiated in 12.7% (14/110) of cases. Most patients (81.6%; 84/103) recovered euthyroidism within 12 months postlobectomy. When excluding patients with autonomous nodule(s), median preoperative TSH was 1.09 (interquartile range [IQR] = 0.70-1.77) mIU/L and 1.80 (IQR = 1.02-2.68) mIU/L in euthyroid and hypothyroid patients, respectively, with multivariate logistic regression confirming the association between an increased preoperative TSH and postlobectomy hypothyroidism (odds ratio = 1.8 [confidence interval 1.08-3.13], p = 0.024). Of the patients who underwent thyroid lobectomy and developed postoperative hypothyroidism (n = 31), 38.7% (12/31) had a preoperative diagnosis of an autonomously functioning thyroid nodule. Conclusions: Thyroid function should be evaluated postlobectomy to assess the need for LT4. LT4 should be considered if the TSH remains elevated, especially if an upward trend is observed or TSH is >10.0 mIU/L. Suppressed preoperative TSH associated with autonomous nodules is an independent risk factor for postlobectomy hypothyroidism.

Thyroid Lobectomy for T1 Papillary Thyroid Carcinoma in Pediatric Patients.

Importance: The current recommendation for pediatric patients with papillary thyroid cancer (PTC) is a total thyroidectomy. This recommendation applies to all stages of PTC, including papillary thyroid microcarcinoma (≤1 cm, T1a) tumors. Objective: To evaluate the characteristics of American Joint Committee on Cancer T1 PTC tumors in a large pediatric population and to identify a subgroup of patients who may benefit from a thyroid lobectomy instead of a total thyroidectomy. Design, Setting, and Participants: This retrospective cohort study was conducted from January 1, 2009, to May 31, 2020. The study took place at a tertiary care medical center and included 102 patients who were surgically treated for T1 PTC: 52 with stage T1a (≤1 cm) tumors and 50 with stage T1b (>1 cm but ≤2 cm) tumors. Main Outcomes and Measures: Primary outcomes included the presence of bilateral disease and lymph node metastasis. Results: A total of 102 patients (mean age, 15.3 years [range, 9.7-18.9 years]; 84 girls [82.4%]) were included in the analysis. Among 52 patients with T1a tumors, 10 (19.2%) had bilateral disease, and 15 (28.8%) had central neck lymph node (N1a) metastasis. Among 50 patients with T1b tumors, 10 (20%) had bilateral and 13 (26%) had N1a disease. Of those with T1a, unilateral multifocality was associated with bilateral disease (odds ratio [OR], 2.1; 95% CI, 1.3-3.4) and N1a disease (OR, 5.1; 95% CI, 1.5-17.6). Both N1a disease (OR, 20.0; 95% CI, 3.5-115.0) and ≥4 positive lymph nodes (OR, 8.6; 95% CI, 1.2-60.9) were associated with bilateral disease. In patients with no pathologic evidence of lymph node metastasis (N0), there was a 95% rate of unilateral PTC. In patients with T1b tumors, unilateral multifocality was also associated with bilateral disease (OR, 1.8; 95% CI, 1.3-2.7). Patients with T1b tumors had an increased risk of lateral (N1b) neck lymph node metastasis when compared with those with T1a tumors (OR, 3.7; 95% CI, 1.0-14.5). Conclusions and Relevance: The findings of this cohort study suggest that, in patients with unifocal T1a PTC without clinically evident nodal disease on preoperative ultrasonography, a thyroid lobectomy and central neck dissection may be considered. If there is no evidence of unilateral multifocality or if there are fewer than 4 positive lymph nodes on postoperative pathology, then close observation may be considered. These findings have substantial clinical implications and may result in practice changes regarding the extent of thyroid surgery on low-stage pediatric PTC.

Utility of Fine-Needle Aspirations to Diagnose Pediatric Thyroid Nodules.

INTRODUCTION: Risk of malignancy for pediatric thyroid nodules classified according to The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) is not well defined. Correlations between risk of malignancy and ancillary clinical data remain inconclusive. We report a single institutional experience of fine-needle aspiration (FNA) to improve upon current management paradigm of thyroid nodules. METHODS: A retrospective chart review of 575 thyroid nodules was performed of 324 patients who underwent 340 FNAs between 2008 and 2018 at the Children's Hospital of Philadelphia. Demographics, ultrasound (US) characteristics, FNA cytology, surgical pathology, and ancillary data were reviewed. RESULTS: The rate of malignancy according to TBSRTC was 0.0% for category I, 0.8% for category II, 15.6% for category III, 54.5% for category IV, 100.0% for category V, and 100.0% for category VI. The cumulative Thyroid Imaging Reporting and Data System (TI-RADS) score was significantly correlated with benign and malignant nodules on pathology (p < 2.2e-16). Distribution of TI-RADS for cytologically indeterminate nodules with benign or malignant pathology revealed significant differences for composition (p = 3.20e-8) and echogenic foci (p = 0.005) but not for echogenicity (p = 0.445), shape (p = 0.160), margins (p = 0.220), and size (p = 0.105). Distributions of thyroid-stimulating hormone levels between benign and malignant patients was significant (p = 1.58e-3). CONCLUSIONS: Nodules with TI-RADS scores >3 should undergo FNA, irrespective of size; surgical resection is recommended for nodules classified as TBSRTC category IV and V due to high risk of malignancy. US surveillance instead of FNA can be performed for nodules with TI-RADS scores ≤3.

Oncogene-specific inhibition in the treatment of advanced pediatric thyroid cancer.

Papillary thyroid cancer (PTC) is the most common form of differentiated thyroid cancer in the pediatric population and represents the second most common malignancy in adolescent females. Historically, PTC has been classified on the basis of histology, however, accumulating data indicate that molecular subtyping based on somatic oncogenic alterations along with gene expression profiling can better predict clinical behavior and may provide opportunities to incorporate oncogene-specific inhibitory therapy to improve the response to radioactive iodine (RAI). In this issue of the JCI, Y.A. Lee, H. Lee, and colleagues showed that oncogenic fusions were more commonly associated with invasive disease, increased expression of MAPK signaling pathway genes (ERK score), and decreased expression of the sodium-iodine symporter, which was restored by RET- and NTRK-inhibitory therapy. These findings lend credence to the idea of reclassifying pediatric thyroid cancers using a three-tiered system, rather than the two-tiered adult system, and open avenues for the treatment of progressive, RAI-refractory PTC in patients.

Development of Novel Follicular Thyroid Cancer Models Which Progress to Poorly Differentiated and Anaplastic Thyroid Cancer.

Recent developments in thyroid cancer research have been hindered by a lack of validated in vitro models, allowing for preclinical experimentation and the screening of prospective therapeutics. The goal of this work is to develop and characterize three novel follicular thyroid cancer (FTC) cell lines developed from relevant animal models. These cell lines recapitulate the genetics and histopathological features of FTC, as well as progression to a poorly differentiated state. We demonstrate that these cell lines can be used for a variety of in vitro applications and maintain the potential for in vivo transplantation into immunocompetent hosts. Further, cell lines exhibit differing degrees of dysregulated growth and invasive behavior that may help define mechanisms of pathogenesis underlying the heterogeneity present in the patient population. We believe these novel cell lines will provide powerful tools for investigating the molecular basis of thyroid cancer progression and lead to the development of more personalized diagnostic and treatment strategies.

Decoding the epitranscriptional landscape from native RNA sequences.

Traditional epitranscriptomics relies on capturing a single RNA modification by antibody or chemical treatment, combined with short-read sequencing to identify its transcriptomic location. This approach is labor-intensive and may introduce experimental artifacts. Direct sequencing of native RNA using Oxford Nanopore Technologies (ONT) can allow for directly detecting the RNA base modifications, although these modifications might appear as sequencing errors. The percent Error of Specific Bases (%ESB) was higher for native RNA than unmodified RNA, which enabled the detection of ribonucleotide modification sites. Based on the %ESB differences, we developed a bioinformatic tool, epitranscriptional landscape inferring from glitches of ONT signals (ELIGOS), that is based on various types of synthetic modified RNA and applied to rRNA and mRNA. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from Escherichiacoli, yeast, and human cells, using either unmodified in vitro transcription RNA or a background error model, which mimics the systematic error of direct RNA sequencing as the reference. The well-known DRACH/RRACH motif was localized and identified, consistent with previous studies, using differential analysis of ELIGOS to study the impact of RNA m6A methyltransferase by comparing wild type and knockouts in yeast and mouse cells. Lastly, the DRACH motif could also be identified in the mRNA of three human cell lines. The mRNA modification identified by ELIGOS is at the level of individual base resolution. In summary, we have developed a bioinformatic software package to uncover native RNA modifications.

The Thyroid Tumor Microenvironment: Potential Targets for Therapeutic Intervention and Prognostication.

Thyroid cancer is the most common endocrine malignancy and incidences are rising rapidly, in both pediatric and adult populations. Many thyroid tumors are successfully treated which results in low mortality rates, but there is often a significant morbidity associated with thyroid cancer treatments. For patients with tumors that are not successfully treated with surgical resection or radioactive iodine treatment, prognosis is dramatically reduced. Patients diagnosed with anaplastic thyroid cancer face a very grim prognosis with a median survival of 6 months post-diagnosis. There is a critical need to identify patients who are at greatest risk of developing persistent disease and progressing to poorly differentiated or anaplastic disease. Furthermore, development of treatments associated with less morbidity would represent a significant improvement for thyroid cancer survivors. It is well established the stromal cells and components of the tumor microenvironment can drive tumor progression and resistance to therapy. Here we review the current state of what is known regarding the thyroid tumor microenvironment and how these factors may contribute to thyroid tumor pathogenesis. Study of the tumor microenvironment within thyroid cancer is a relatively new field, and more studies are needed to dissect the complex and dynamic crosstalk between thyroid tumor cells and its tumor niche.

miRNA expression can classify pediatric thyroid lesions and increases the diagnostic yield of mutation testing.

BACKGROUND: Genetic alterations in multiple cell signaling pathways are involved in the molecular pathogenesis of thyroid cancer. Oncogene mutation testing and gene-expression profiling are routinely used for the preoperative risk management of adult thyroid nodules. In this study, we evaluated the potential value of miRNA biomarkers for the classification of pediatric thyroid lesions. PROCEDURE: Double-blind case-control study with 113 resected pediatric lesions: 66 malignant and 47 benign. Quantitative and qualitative molecular data generated with a 10-miRNA expression panel (ThyraMIR) and a next-generation sequencing oncogene panel (ThyGeNEXT) were compared with clinicopathological parameters. RESULTS: miRNAs were differentially expressed in benign versus malignant tumors with distinct expression patterns in different histopathology categories. The 10-miRNA classifier identified 39 (59%) malignant lesions with 100% specificity. A positive classifier score was associated with lymph node metastasis, extrathyroidal extension and intrathyroidal spread. Genetic alterations associated with increased risk for malignancy were detected in 35 (53%) malignant cases, 20 positive for point mutations in BRAF, HRAS, KRAS, NRAS, PIK3CA, or TERT and 15 positive for gene rearrangements involving ALK, NTRK3, PPARG, or RET. The 10-miRNA classifier correctly identified 11 mutation-negative malignant cases. The performance of the combined molecular test was 70% sensitivity and 96% specificity with an area under the curve of 0.924. CONCLUSIONS: These data suggest that the regulatory miRNA pathways underlying thyroid tumorigenesis are similar in adults and children. miRNA expression can identify malignant lesions with high specificity, augment the diagnostic yield of mutation testing, and improve the molecular classification of pediatric thyroid nodules.