Transcriptomic Analysis Reveals Dysregulation of the Mycobiome and Archaeome and Distinct Oncogenic Characteristics according to Subtype and Gender in Papillary Thyroid Carcinoma.

Papillary Thyroid Carcinoma (PTC) is characterized by unique tumor morphology, treatment response, and patient outcomes according to subtype and gender. While previous studies have implicated the intratumor bacterial microbiome in the incidence and progression of PTC, few studies have investigated the potential role of fungal and archaeal species in oncogenesis. In this study, we aimed to characterize the intratumor mycobiome and archaeometry in PTC with respect to its three primary subtypes: Classical (CPTC), Follicular Variant (FVPTC), and Tall Cell (TCPTC), and also with respect to gender. RNA-sequencing data were downloaded from The Cancer Genome Atlas (TCGA), including 453 primary tumor tissue samples and 54 adjacent solid tissue normal samples. The PathoScope 2.0 framework was used to extract fungal and archaeal microbial read counts from raw RNA-sequencing data. Overall, we found that the intratumor mycobiome and archaeometry share significant similarities in CPTC, FVPTC, and TCPTC, although most dysregulated species in CPTC are underabundant compared to normal. Furthermore, differences between the mycobiome and archaeometry were more significant between males and females, with a disproportionate number of fungal species overabundant in female tumor samples. Additionally, the expression of oncogenic PTC pathways was distinct across CPTC, FVPTC, and TCPTC, indicating that these microbes may uniquely contribute to PTC pathogenesis in each subtype. Furthermore, differences in the expression of these pathways were observed between males and females. Finally, we found a specific panel of fungi to be dysregulated in BRAF V600E-positive tumors. This study demonstrates the potential importance of microbial species to PTC incidence and oncogenesis.

Relationship between genetic alterations and clinicopathological characteristics of papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is characterized by proliferation of follicular cells with distinctive nuclear features such as ground glass appearance, nuclear groove and pseudoinclusion. From the proliferation pattern, PTC is divided into several histological subtypes; conventional histology is classified as papillary type, and there are also follicular and solid variants. PTC is heterogeneous in genetic alterations. PTC with BRAF mutation presents a histology of conventional PTC, and follows an aggressive clinical course. Most cases of PTC with RAS mutation show a follicular variant, and prognosis is favorable. RET/PTC1 is observed sporadically and in young cases, and prognosis is favorable. RET/PTC3 is associated with radiation exposure, and the solid variant is frequent. ETV6-NTRK3 may be associated with radiation exposure, and the clinical course is aggressive. Mutation in the telomerase reverse transcriptase promoter is observed in PTC cases involving elderly male patients. Tumor size is large, associated with distant metastasis and advanced stage. This mutation is found concomitantly with BRAF mutation, and the clinical course is aggressive. Genetic alterations form subsets of PTC with distinct clinicopathological features. Careful assessment of clinicopathological features is considered useful in predicting clinical course and when planning treatment.

Role of molecular markers to predict distant metastasis in papillary thyroid carcinoma: Promising value of TERT promoter mutations and insignificant role of BRAF mutations-a meta-analysis.

The presence of distant metastasis is associated with an adverse outcome in papillary thyroid cancer. We performed a meta-analysis to investigate the role of molecular markers as predictors for distant metastasis in papillary thyroid cancer. Four electronic databases including PubMed, Web of Science, Scopus, and Virtual Health Library were searched, and odds ratio and its 95% confidence interval concerning the association of BRAF, RAS, and TERT promoter mutations and RET/PTC rearrangements with distant metastasis were calculated using random-effects model. In total, 42 studies with 11,109 papillary thyroid cancers were included for meta-analyses. Overall, the presence of TERT promoter (odds ratio = 5.95; 95% confidence interval = 2.95-11.99), RAS mutations (odds ratio = 2.5; 95% confidence interval = 1.00-6.22), and RET/PTC rearrangements (odds ratio = 1.92; 95% confidence interval = 1.03-3.56) were found to be associated with a significantly increased risk for distant metastasis. BRAF mutations were not associated with an elevated risk for distant metastasis (odds ratio = 0.79; 95% confidence interval = 0.54-1.16). In conclusion, our study demonstrated the promising value of few molecular biomarkers, especially TERT promoter mutations in predicting distant metastasis in papillary thyroid cancers, while BRAF mutations showed no association with distant metastasis. Our study affirms the value of selected mutations for tumor risk stratification and assessment of patients' prognosis.

The Genetics of Papillary Microcarcinomas of the Thyroid: Diagnostic and Prognostic Implications.

Papillary microcarcinoma of the thyroid (mPTC) is defined by the WHO as a papillary thy-roid cancer measuring 10mm or less in diameter and it is nowadays a topic of intense debate among the members of the medical community due to its apparent "epidemic" rise. Although these tumors follow almost always an indolent clinical course and carry an excellent prognosis, it is known that a small sub-set may display a potentially aggressive behavior. Nevertheless, we still lack an accurate way of predict-ing those which will cause significant disease. In an attempt to address this problem, a number of clini-co-pathologic features have been studied as poor prognostic markers in mPTC, and their association with known genetic alterations in thyroid cancer has been evaluated. Herein we review the present knowledge concerning mPTC's genetic profile, namely the prevalence of BRAF (V600E), RAS and TERT promoter mutations and RET/PTC and PAX8-PPARG rearrangements and report the results of the evaluation in the putative prognostic value of these genetic alterations in mPTC.

Diagnostic Limitation of Fine-Needle Aspiration (FNA) on Indeterminate Thyroid Nodules Can Be Partially Overcome by Preoperative Molecular Analysis: Assessment of RET/PTC1 Rearrangement in BRAF and RAS Wild-Type Routine Air-Dried FNA Specimens.

Molecular markers are helpful diagnostic tools, particularly for cytologically indeterminate thyroid nodules. Preoperative RET/PTC1 rearrangement analysis in BRAF and RAS wild-type indeterminate thyroid nodules would permit the formulation of an unambiguous surgical plan. Cycle threshold values according to the cell count for detection of the RET/PTC1 rearrangement by real-time reverse transcription-polymerase chain reaction (RT-PCR) using fresh and routine air-dried TPC1 cells were evaluated. The correlation of RET/PTC1 rearrangement between fine-needle aspiration (FNA) and paired formalin-fixed paraffin-embedded (FFPE) specimens was analyzed. RET/PTC1 rearrangements of 76 resected BRAF and RAS wild-type classical PTCs were also analyzed. Results of RT-PCR and the Nanostring were compared. When 100 fresh and air-dried TPC1 cells were used, expression of RET/PTC1 rearrangement was detectable after 35 and 33 PCR cycles, respectively. The results of RET/PTC1 rearrangement in 10 FNA and paired FFPE papillary thyroid carcinoma (PTC) specimens showed complete correlation. Twenty-nine (38.2%) of 76 BRAF and RAS wild-type classical PTCs had RET/PTC1 rearrangement. Comparison of RET/PTC1 rearrangement analysis between RT-PCR and the Nanostring showed moderate agreement with a κ value of 0.56 (p = 0.002). The RET/PTC1 rearrangement analysis by RT-PCR using routine air-dried FNA specimen was confirmed to be technically applicable. A significant proportion (38.2%) of the BRAF and RAS wild-type PTCs harbored RET/PTC1 rearrangements.

Diffuse sclerosing variant of papillary thyroid carcinoma: major genetic alterations and prognostic implications.

AIM: Diffuse sclerosing variant of papillary thyroid carcinoma (DSV-PTC) is an uncommon variant of PTC, and its prognostic significance remains controversial. The aim of this study was to investigate the major genetic alterations of DSV-PTC and their prognostic implications. METHODS AND RESULTS: We included 37 patients with DSV-PTC who underwent thyroid surgery and had formalin-fixed paraffin-embedded samples. We tested for a panel of genetic alterations, including BRAF(V) (600E) , NRAS codon 61, HRAS codon 12/13/61 and KRAS codon 12/13 point mutations as well as RET/PTC1, RET/PTC3 and PAX8/PPARγ rearrangements using reverse transcription real-time polymerase chain reaction (PCR). All genetic alterations found on PCR were confirmed by Sanger sequencing. Associations between the identified genetic alterations and clinicopathological characteristics were evaluated. Among 37 cases of DSV-PTC, 17 were positive for RET/PTC1 (46%), six for RET/PTC3 (16%) and nine for BRAF(V) (600E) (24%). All mutations/rearrangements were mutually exclusive. The remaining five cases had none of the above genetic alterations. DSV-PTC with RET/PTC3 rearrangement was associated with advanced-stage disease, including T4 and distant metastasis (P < 0.05). Patients with RET/PTC3 showed a higher frequency of persistent disease (P < 0.01). In contrast, DSV-PTC with RET/PTC1 was associated with a higher prevalence of disease remission (P < 0.05) and coexistent Hashimoto's thyroiditis (P < 0.01). CONCLUSION: Taken together, RET/PTC rearrangement was the major genetic alteration seen in patients with DSV-PTC, and the RET/PTC3 rearrangement was associated with advanced stage at diagnosis and poor clinical outcome.

Laser capture microdissection is a valuable tool in the preoperative molecular screening of follicular lesions of the thyroid: an institutional experience.

OBJECTIVES: The application of molecular tests to thyroid fine needle aspiration (FNA) has been shown to be a valuable tool to better refine the pre-operative malignant risk of patients with indeterminate cytology results. In this study, we investigated the feasibility of using the laser capture microdissection (LCM) technique to obtain DNA and RNA for molecular tests in routine thyroid FNA smears. METHODS: Nine coupled FNA and histological retrospective cases and 31 prospective FNA cases with a follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) diagnosis were included in this study. Both cytological and histological specimens were investigated by direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) for BRAF and RAS mutations and for PAX8/PPARG and RET/PTC rearrangements, respectively. RESULTS: LCM yielded good DNA and RNA quality in all cases (100%) in both series, irrespective of the staining used (Giemsa, Papanicolaou, immunostain for thyroglobulin) and the cytology technique (conventional or liquid-based preparations). Total mutations found in the FNA and in the corresponding histological specimen in both series were: one PAX8/PPARG rearrangement in a follicular carcinoma (FC), four NRAS mutations [in two FCs, one papillary carcinoma and one follicular adenoma (FA)] and one HRAS mutation in one FA. The sensitivity was 67% and the specificity was 91%. CONCLUSIONS: LCM is a valuable tool to obtain good quality DNA and RNA for molecular tests in cytological material from thyroid FNA, and can be a useful option in the management of patients with an FN/SFN FNA diagnosis.

BRAF mutation correlates with recurrent papillary thyroid carcinoma in Chinese patients.

PURPOSE: We investigated correlations of somatic BRAF V600E mutation and RET/PTC1 rearrangement with recurrent disease in Chinese patients with papillary thyroid carcinoma (ptc). METHODS: This prospective study included 214 patients with ptc histologically confirmed between November 2009 and May 2011 at a single institute. RESULTS: We found somatic BRAF V600E mutation in 68.7% and RET/PTC1 rearrangement in 25.7% of the patients. Although BRAF mutation was not significantly associated with clinicopathologic features such as patient sex or age, multicentric disease, thyroid capsule invasion, tumour stage, or nodal metastasis, it was significantly associated with recurrent disease. Multivariate analysis revealed that BRAF mutation and tumour size were independent risk factors associated with recurrent disease, with odds ratios of 9.072 and 2.387 respectively. The area under the receiver operating characteristic curve increased 8.3% when BRAF mutation was added to the traditional prognostic factors, but that effect was statistically nonsignificant (0.663 vs. 0.746, p = 0.124). RET/PTC1 rearrangement and nodal metastasis were significantly associated in all patients (p = 0.042), marginally associated in ptc patients (p = 0.051), but not associated in microptc patients (p = 0.700). RET/PTC1 rearrangement was not significantly associated with recurrent disease. CONCLUSIONS: BRAF positivity is an independent predictor of recurrent disease in ptc.

Application value of multi-gene mutation detection in the clinical management of pediatric papillary thyroid carcinoma: a preliminary exploration.

Objectives: Thyroid cancer rarely occurs in children and adolescents. Molecular markers such as BRAF, RAS, and RET/PTC have been widely used in adult PTC. It is currently unclear whether these molecular markers have equivalent potential for application in pediatric patients. This study aims to explore the potential utility of a multi-gene conjoint analysis based on next-generation targeted sequencing for pediatric papillary thyroid carcinoma (PTC). Materials and methods: The patients diagnosed with PTC (aged 18 years or younger) in the pediatrics department of Lishui District Hospital of Traditional Chinese Medicine were retrospectively screened. A targeted enrichment and sequencing analysis of 116 genes associated with thyroid cancer was performed on paraffin-embedded tumor tissues and paired paracancerous tissue of fifteen children (average age 14.60) and nine adults (average age 49.33) PTC patients. Demographic information, clinical indicators, ultrasonic imaging information and pathological data were collected. The Kendall correlation test was used to establish a correlation between molecular variations and clinical characteristics in pediatric patients. Results: A sample of 15 pediatric PTCs revealed a detection rate of 73.33% (11/15) for driver gene mutations BRAF V600E and RET fusion. Compared to adult PTCs, the genetic mutation landscape of pediatric PTCs was more complex. Six mutant genes overlap between the two groups, and an additional seventeen unique mutant genes were identified only in pediatric PTCs. There was only one unique mutant gene in adult PTCs. The tumor diameter of pediatric PTCs tended to be less than 4cm (p<0.001), and the number of lymph node metastases was more than five (p<0.001). Mutations in specific genes unique to pediatric PTCs may contribute to the onset and progression of the disease by adversely affecting hormone synthesis, secretion, and action mechanisms, as well as the functioning of thyroid hormone signaling pathways. But, additional experiments are required to validate this hypothesis. Conclusion: BRAF V600E mutation and RET fusion are involved in the occurrence and development of adolescent PTC. For pediatric thyroid nodules that cannot be determined as benign or malignant by fine needle aspiration biopsy, multiple gene combination testing can provide a reference for personalized diagnosis and treatment by clinical physicians.

Coexisting RET/PTC and TERT Promoter Mutation Predict Poor Prognosis but Effective RET and MEK Targets in Thyroid Cancer.

PURPOSE: To investigate the role of coexisting RET/PTC rearrangement and TERT promoter mutation in the prognosis and therapeutic targeting in papillary thyroid cancer (PTC). METHODS: A total of 669 PTC patients with complete clinical follow-up and genetic data were pooled from thyroid cancer datasets TCGA, MSK MetTropism, and MSK-IMPACT, from whom 163 patients (112 women and 47 men, 4 unknown) with wild-type BRAF/RAS were identified, with median age (IQR) of 46.00 (33.00, 61.00) years and median follow-up time (IQR) of 16.13 (8.09, 27.91) months for comparative genotype cohort analysis of mortality. RESULTS: There was a significant concurrence index between RET/PTC and TERT promoter mutations, being 2.040 (95% CI 1.110-3.747, P = 0.023). Mortality occurred in 5/100 (5%) patients harboring neither mutation, 2/18 (11.1%) patients harboring TERT promoter mutation alone, 0/31 (0%) patients harboring RET/PTC alone, and 7/14 (50%) patients harboring both genetic alterations, corresponding to HRs (95% CI) of 1 (Reference), 2.469 (0.405-14.02), 3.296e-09 (0-inf), and 9.019 (2.635-30.87), respectively, which remained essentially unchanged after adjustment for patient race, sex, and age. Similar results were observed with BRAF/RAS and TERT promoter mutations. Mechanistically, RET/PTC used the MAP kinase pathway to upregulate the mutated TERT, but not the wild-type TERT, and, correspondingly, targeting RET and MEK could suppress mutated TERT but not the wild-type TERT. CONCLUSION: Coexisting RET/PTC and TERT promoter mutation identify PTC as a unique clinical entity with high mortality, providing new implications for genetic-based prognostication and potential therapeutic targeting of RET and MEK guided by RET/PTC and TERT status.

A study to evaluate association of nuclear grooving in benign thyroid lesions with RET/PTC1 and RET/PTC3 gene translocation.

INTRODUCTION: Papillary thyroid carcinoma (PTC) is the most common malignant lesion of the thyroid characterized by unique histological features like nuclear grooving, nuclear clearing, and intra-nuclear inclusions. However, nuclear grooves are observed even in benign thyroid lesions (BTL) like nodular goiter (NG), Hashimoto's thyroiditis (HT), and follicular adenoma (FA) resulting in diagnostic dilemma of the presence of PTC in such BTL. RET/PTC gene translocation is one of the most common oncogenic rearrangements seen in PTC, known to be associated with nuclear grooving. Among different types of RET/PTC translocations, RET/PTC1 and RET/PTC3 gene translocations are the most common types. These translocations have also been identified in many BTL like hyperplastic nodules and HT. Our study aimed to determine the frequency of nuclear grooving in BTL and evaluate their association with RET/PTC1 and RET/PTC3 gene translocation. METHODS: Formalin-fixed, paraffin-embedded (FFPE) tissue blocks of NG, HT, and FA were included in the study. The hematoxylin and eosin (H&E) stained sections were evaluated for the presence of nuclear grooving/high power field (hpf) and a scoring of 0 to 3 was used for the number of grooves. Sections of 10 µ thickness were cut and the cells containing the nuclear grooves were picked using Laser-Capture microdissection. About 20 to 50 such cells were microdissected in each of the cases followed by RNA extraction, cDNA conversion, realtime-polymerase chain reaction (RQ-PCR) for RET/PTC1 and RET/PTC3 gene translocation, and the findings were analyzed for statistical significance. RESULTS: Out of 87 BTL included in the study, 67 (77.0%) were NG, 12 (13.7%) were HT, and 8 (9.2%) were FA. Thirty-two cases (36.8%) had nuclear grooving with 18 out of 67 NG, 6 out of 12 HT, and all 8 cases of FA showing a varying number of nuclear grooves. A significant association between the number of nuclear grooves with RET/PTC gene translocation (p-value of 0.001) was obtained. A significant association of HT with RET/PTC gene translocation (p-value of 0.038) was observed. RET/PTC1 and RET/PTC3 translocation were seen in 5 out of 87 cases, with HT showing positivity in 2 and FA in 1 case for RET/PTC1 and HT in 1 and FA in 2 cases for RET/PTC3 gene translocation with 1 case of FA being positive for both RET/PTC1 and RET/PTC3 gene translocation. CONCLUSIONS: The frequency of nuclear grooving among BTLs in our study was 36.8%. Our study shows, that when BTLs, show nuclear grooves, with an increase in the nuclear size, oval and elongated shape, favors the possibility of an underlying genetic aberration like RET/PTC gene translocation, which in turn supports the reporting pathologist to suggest a close follow up of the patients on seeing such nuclear features on cytology or histopathology sample, particularly in HT.

Nodal Metastases Associated With Fusion Oncogenes Are Age Dependent in Young Adult Patients With Thyroid Cancer.

CONTEXT: Fusion oncogenes, especially those involving RET or NTRK, are known drivers of papillary thyroid cancer (PTC). They are prevalent in pediatric patients and correlate with aggressive tumor behavior. OBJECTIVE: We explored the age dependence of fusion oncogenes and aggressive tumor behavior in young adult PTC patients. EXPERIMENTAL DESIGN: We examined 150 tumors from 142 PTC patients aged between 17∼35 years old with established tumor-node-metastasis stages. Oncogenic drivers and the thyroid differentiation score (TDS) were determined by DNA and RNA sequencing of a target panel. Transcriptome analysis was performed in PTCs with RET fusions. RESULTS: Among 150 PTCs, we detected BRAF V600E (n = 105), RET fusions (n = 15), NTRK3 fusions (n = 8), and BRAF fusions (n = 4). We found that fusion oncogenes were associated with nodal metastasis when age was tiered into 3 groups: <25 years, 25∼29 years, and 30∼35 years. Patients under 25 years old showed a marginal increase in tumor stage compared to those over 25 years (75.00% vs 21.74%, P = .0646). Risk of lateral lymph node metastasis increased with younger age (75.00% vs 27.27% vs 8.33%, P = .0369). As with advanced tumor and node stage, patients harboring fusion oncogenes and aged under 25 years showed the lowest TDS; genes associated with immunoglobulin production and production of molecular mediators of the immune response were significantly upregulated. CONCLUSIONS: Adult PTC patients under 25 years with fusion oncogenes showed a tendency toward advanced tumor stage and lower thyroid differentiation. Integrating onset age together with oncogenic alterations is worthwhile when managing adult PTC patients.

The Difference in Clinical Behavior of Gene Fusions Involving RET/PTC Fusions and THADA/IGF2BP3 Fusions in Thyroid Nodules.

BACKGROUND: Molecular testing has been used as an adjunct to morphological evaluation in the workup of thyroid nodules. This study investigated the impact of two gene fusions, RET/PTC and THADA/IGF2BP3, that have been described as oncogenic events in thyroid neoplasms. METHODS: We performed a retrospective, single-centered study at a McGill University teaching hospital in Montreal, Canada, from January 2016 to August 2021. We included patients who underwent surgery for thyroid nodules that pre-operatively underwent molecular testing showing either RET/PTC or THADA/IGF2BP3 gene fusion. RESULTS: This study included 697 consecutive operated thyroid nodules assessed using molecular testing, of which five had the RET/PTC fusion and seven had the THADA/IGF2BP3 fusion. Of the five nodules in the RET/PTC group, 100% were malignant and presented as Bethesda V/VI. Eighty percent (4/5) were found to have lymph node metastasis. Twenty percent (1/5) had extrathyroidal extensions. Sixty percent (3/5) were a diffuse sclerosing variant of papillary thyroid carcinoma, and the rest were the classical variant. Of the seven THADA/IGF2BP3 nodules, all presented as Bethesda III/IV and 71.4% (5/7) were malignant based on the final pathology analysis, and 28.6% (2/7) were NIFTP. All the THADA/IGF2BP3 fusion malignancies were a follicular variant of papillary thyroid carcinoma. None had lymph node metastasis or displayed extrathyroidal extensions. CONCLUSIONS: RET/PTC nodules presented as Bethesda V/VI and potentially had more aggressive features, whereas THADA/IGF2BP3 nodules presented as Bethesda III/IV and had more indolent behavior. This understanding may allow clinicians to develop more targeted treatment plans, such as the extent of surgery and adjuvant radioactive iodine treatment.

RET/PTC rearrangement in papillary thyroid carcinoma arising in malignant struma ovarii with abdominal wall metastasis and cervical thyroid gland: a case report and review of the literature.

BACKGROUND: Struma ovarii refers to rare mature cystic teratomas containing at least 50% of thyroid tissue, and malignant transformation is known to be even rarer. The synchronous development of malignant struma ovarii and cervical thyroid carcinoma are also scarce and poorly understood due to limited data about molecular features. Here, we present the first report of RET/PTC 1 rearrangement in synchronous metastatic malignant struma ovarii to the abdominal wall and cervical thyroid cancer. CASE PRESENTATION: We described a 47-year-old multigravida woman with bilateral adnexal and lower abdominal wall masses detected during the evaluation of abnormal uterine bleeding. The patient underwent a hysterectomy, bilateral salpingo-oophorectomy, and surgical removal of abdominal wall mass. Then, the pathological evaluation revealed papillary thyroid carcinoma (PTC) within struma ovarii and metastatic PTC in the abdominal wall fibro adipose tissue. Further, cervical thyroid gland physical examination and ultrasound illustrated a nodule within the left lobe. Subsequently, a total thyroidectomy was performed, and a histological examination revealed PTC. Furthermore, all affected tissue, i.e., struma ovarii, abdominal wall metastasis, and cervical thyroid gland tested for BRAF and RAS mutations and RET/PTC 1 rearrangement. RET/PTC 1 rearrangement was identified among all three different sites. Finally, after six years of follow-up, the patient had no evidence of recurrence or distant metastasis. CONCLUSIONS: In light of these findings, malignant struma ovarii might yield a clue to cervical thyroid carcinoma, and the molecular analysis could provide valuable information for understanding the underlying mechanism, tumor clinicopathological behaviors, and prognosis.

Update on International Medical Taxonomies of Biomarkers and Their Applications in Management of Thyroid Cancers.

Biomarkers (BMs) are medical signs which can be precisely measured and reproduced. Mainly, BMs provide information on the likely disease which can occur in an individual. On the other hand, BMs also signal disease recurrence in patients receiving therapy. The U.S. Food and Drug Administration coupled with the National Institutes of Health and the European Medicines Agency have proposed two distinct procedures to validate BMs. These agencies have elaborated two glossaries to describe the role of BMs. The aim of this study was to investigate medical taxonomies adopted by different governmental agencies for BM validation. Additional goals were to analyze efficiencies of the validated and candidate BMs for thyroid cancers (TCs). Currently, thyroglobulin is validated for monitoring TCs. Sorafenib-tosylate, Doxorubicin-hydrochloride, Vandetanib, Cabozantinib-s-malate, Dabrafenib-mesylate, Trametinib-dimethyl-sulfoxide, Lenvatinib-mesylate, Pralsetinib and Selpercatinib are validated for TC treatment. Among candidate BMs for TC diagnosis, there are molecular combinations including BRAF, RAS, RET/PTC and PAX8-PPARγ mutations. Noteworthy are BRAF and RET/PTC alterations already validated as targets of Dabrafenib-mesylate, Pralsetinib and Selpercatinib. Finally, cellular expressions of c-met in nodal TC metastases have diagnostic imaging applications. On the basis of this analysis, BM taxonomies should have common standards internationally recognized. BMs show different efficiencies depending on their diagnostic or therapeutic use.

Unique Molecular Signatures Are Associated with Aggressive Histology in Pediatric Differentiated Thyroid Cancer.

Background: Molecular testing (MT) enhances the diagnostic accuracy of thyroid fine-needle aspiration biopsy, reducing the need for diagnostic lobectomy in adult patients with indeterminate nodules (Bethesda class III/IV). However, little is known about genetic alterations in pediatric thyroid carcinoma (TC). Our aim was to analyze MT results of pediatric differentiated TC (DTC) cases to determine associations with histological and clinical features. Methods: A retrospective review identified all patients (aged <19 years) diagnosed with DTC from 2001 to 2017 at the University of Pittsburgh Medical Center. Histology was rereviewed to confirm diagnosis and identify tissue for MT using next-generation sequencing (ThyroSeq, version 3, TSv3). Correlation with histological and clinical features was analyzed using regression analysis. Results: Of 71 patients with MT results, 62 (87%) patients had papillary TC. All patients were alive at a median follow-up of 6 years (range 18 days to 18 years). Genetic alterations were identified in 65 (92%) patients. These alterations were grouped as BRAF-like point mutations or fusions (39, 55%), RAS-like mutations or fusions (21, 30%), or copy number alterations (5, 7%). On multiple regression analysis accounting for patient sex and tumor size in patients with papillary TC, increased tumor stage (ß: 0.234, p < 0.001), multifocal disease (odds ratio [OR]: 3.60, p = 0.042), and lymph node metastases (OR: 6.13, p = 0.044) were associated with BRAF-like gene fusions. When considering individual mutations, ETV6/NTRK3 fusions were associated with increased tumor stage (ß: 2.07, p = 0.023) and BRAF-like point mutations were associated with increased likelihood of surgery for recurrence over time (hazard ratio: 19.5, p = 0.004). Conclusions: Among our cohort of pediatric TC patients who underwent comprehensive MT, >90% had an identifiable genetic alteration. Aggressive features were primarily associated with BRAF-like gene fusions. Preoperative MT results may be useful in guiding the extent of the initial operation in pediatric patients (aged <19 years) with TC.

Clinical-Pathological Features and Treatment Outcome of Patients With Hobnail Variant Papillary Thyroid Carcinoma.

Papillary thyroid carcinoma (PTC) with hobnail areas above 30% is classified as hobnail variant (HVPTC). Although it is widely accepted that HVPTC has a worse outcome than classical PTC, it is unclear whether PTC with hobnail features below 30% is as aggressive as HVPTC. We gathered the largest mono-institutional series of PTC with hobnail areas and HVPTC to evaluate differences in terms of pathological features of aggressiveness, molecular profile, and treatment outcome. A total of 99 PTC with hobnail features above 5% were retrospectively selected; 34 of them met the criteria for HVPTC (0.4% of all PTC diagnosed at our institution). All tumors showed high rates of extra-thyroidal extension (40.4%), lymph node metastasis (68.1% of patients with lymphadenectomy), and vascular emboli (49.5%), with no differences according to the 30% cutoff. On the other hand, distant metastases were present in HVPTC only (9.4%). Also, advanced age, advanced disease stage, and TERT promoter mutation were associated with HVPTC. More than half of the patients with follow-up had structural or biochemical persistence after 1 year from surgery. Structural persistence was significantly more common in patients with HVPTC (37.5% vs. 8.7%), while no differences were observed considering structural and biochemical persistence together. The presence of hobnail features identifies locally aggressive tumors, and, consequently, it should be always acknowledged in the pathological report. However, tumors with more than 30% hobnail areas frequently present TERT promoter mutations, advanced disease stage, and structural persistence after radioiodine ablation.

Mutational analysis using next generation sequencing in pediatric thyroid cancer reveals BRAF and fusion oncogenes are common.

BACKGROUND: We previously described mutation rates of BRAFV600E, RAS, RET-PTC and PAX8-PPARγ in pediatric subjects with well-differentiated thyroid cancer (WDTC). We expanded the cohort adding next-generation sequencing (NGS) and assessed genotype-phenotype correlations. METHODS: Single-center retrospective cohort examining thyroidectomy tissue blocks from consecutive pediatric WDTC patients between 2001 and 2015. Tissues were analyzed at Quest Diagnostics for BRAF, RAS mutations, RET-PTC and PAX8-PPARγ, and additional fusions, using standalone and NGS tests. WDTC included papillary (PTC), follicular (FTC) and follicular-variant PTC (FVPTC). RESULTS: We genotyped 46 samples (36 females). Mean age at diagnosis was 14.7 years and the cohort comprised of mostly Hispanic (60.9%) and Caucasian (26.1%) patients. Mean follow-up was 3.5 years. Genetic alterations (GA) were noted in 69.6%, with BRAFV600E (n = 11), and RET-PTC (n = 8) detected only in PTC. GA were detected in 2/7 FTC (1 PAX8-PPARγ, 1 NRAS) and 6/10 FVPTC (3 PAX8-PPARγ, 1 STRN-ALK, 1 BRAFK601E, 1 NRAS). Patients with BRAFV600E were predominantly Hispanic (81.8%) and >15 years (81.8%), whereas 87.5% RET-PTC and 50% other-fusions occurred in patients ≤15 years (p = 0.044). Of the 29 PTC patients, 82.8% had GA: BRAFV600E (37.9%), RET-PTC (27.6%), 17.2% other fusion-oncogenes (2 -ALK, 3 -NTRK). Non-RET fusions had the highest vascular invasion (100%, p = 0.042 vs RET-PTC) and frequent lymphatic invasion (80%). GA were most common in PTC with cervical metastasis. CONCLUSIONS: BRAFV600E was the most common single mutation, especially in older and Hispanic adolescents. All fusions combined are more common than BRAFV600E. NGS reveals a genetic basis in most pediatric WDTC, which may have implications for the role of molecular testing and systemic therapy.

Emerging Role of Oxidative Stress on EGFR and OGG1-BER Cross-Regulation: Implications in Thyroid Physiopathology.

Thyroid diseases have a complex and multifactorial aetiology. Despite the numerous studies on the signals referable to the malignant transition, the molecular mechanisms concerning the role of oxidative stress remain elusive. Based on its strong oxidative power, H2O2 could be responsible for the high level of oxidative DNA damage observed in cancerous thyroid tissue and hyperactivation of mitogen-activated protein kinase (MAPK) and PI3K/Akt, which mediate ErbB signaling. Increased levels of 8-oxoG DNA adducts have been detected in the early stages of thyroid cancer. These DNA lesions are efficiently recognized and removed by the base excision repair (BER) pathway initiated by 8-oxoG glycosylase1 (OGG1). This study investigated the relationships between the EGFR and OGG1-BER pathways and their mutual regulation following oxidative stress stimulus by H2O2 in human thyrocytes. We clarified the modulation of ErbB receptors and their downstream pathways (PI3K/Akt and MAPK/ERK) under oxidative stress (from H2O2) at the level of gene and protein expression, according to the mechanism defined in a human non-pathological cell system, Nthy-ori 3-1. Later, on the basis of the results obtained by gene expression cluster analysis in normal cells, we assessed the dysregulation of the relationships in a model of papillary thyroid cancer with RET/PTC rearrangement (TPC-1). Our observations demonstrated that a H2O2 stress may induce a physiological cross-regulation between ErbB and OGG1-BER pathways in normal thyroid cells (while this is dysregulated in the TPC-1 cells). Gene expression data also delineated that MUTYH gene could play a physiological role in crosstalk between ErbB and BER pathways and this function is instead lost in cancer cells. Overall, our data on OGG1 protein expression suggest that it was physiologically regulated in response to oxidative modulation of ErbB, and that these might be dysregulated in the signaling pathway involving AKT in the progression of thyroid malignancies with RET/PTC rearrangements.

An updated patent review of rearranged during transfection (RET) kinase inhibitors (2016-present).

INTRODUCTION: Rearranged during transfection (RET) is a transmembrane receptor tyrosine kinase. Aberrations in RET signaling due to mutations, gene fusions, or overexpression can lead to carcinomas. Six inhibitors have been approved for the treatment of RET-driven cancers: vandetanib, cabozantinib, lenvatinib, sorafenib, selpercatinib, and pralsetinib. Only selpercatinib and pralsetinib have been developed specifically for RET, while the remaining are multikinase inhibitors. Several other RET targeted candidates are under clinical development. AREAS COVERED: This review covers recent patent literature describing small molecules that are active against RET since 2016 till present. EXPERT OPINION: RET represents a major therapeutic target as its alterations occur in nearly 2% of all cancers. Recent approvals for RET targeted therapy have been developed specifically to target the RET oncogene. These approvals represent a paradigm shift from the last decade to now focus on the development of selective RET inhibitors rather than multikinase inhibitors. These newly approved RET inhibitors still have clinical issues with drug resistance. It is imperative that the next iteration of RET inhibitors are developed to block common treatment-resistant mutations. To accomplish this, RET inhibitors should be developed in concert with genomic profiling to ensure the most relevant clinical mutations are targeted.