Thyroid Cancer Samples Research Articles

FTO/IGF2BP2-mediated N6 methyladenosine modification in invasion and metastasis of thyroid carcinoma via CDH12.

Epigenetic reprogramming plays a critical role in cancer progression of cancer, and N6-methyladenosine (m6A) is the most common RNA modification in eukaryotes. The purpose of this study was to explore the related modification mode of m6A regulator construction and evaluate the invasion and migration of thyroid cancer. Our results showed that m6A levels were significantly increased in papillary thyroid cancer (PTC) and anaplastic thyroid cancer (ATC) samples, which may have been induced by the down-regulation of demethylase fat mass and obesity-associated gene (FTO). Moreover, FTO inhibited PTC and ATC invasion and metastasis through the epithelial-to-mesenchymal transition (EMT) pathway in vivo and in vitro. Mechanistically, an m6A-mRNA epitranscriptomic microarray showed that Cadherin 12 (CDH12) is the key target gene mediated by FTO in an m6A-dependent manner. CDH12 promotes invasion and metastasis through the EMT pathway in thyroid cancer, both in vivo and in vitro. Furthermore, we found that insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is an important m6A reading protein, that regulates the stability of CDH12 mRNA and mediates EMT progression, thereby promoting the invasion and metastasis of PTC and ATC. Thus, FTO, IGF2BP2 and CDH12 may be effective therapeutic targets for PTC and ATC with significant invasion or distant metastasis. Schematic summary of FTO-IGF2BP2 axis in modulation of CDH12 mRNA m6A and upregulation of CDH12 expression in the invasion and metastasis of thyroid carcinoma.

6885 Glucose Utilization Differs Between RAS-like and BRAF-like Thyroid Cancer

Abstract Disclosure: S. Kumari: None. E. Chuki: None. J. Klubo-Gwiezdzinska: None. Background: RAS-like thyroid cancer (TC) tends to be more differentiated and less [1][8]Fluorodeoxyglucose Positron Emission Tomography-avid as compared with BRAF-like TC. Therefore, we hypothesized that glucose metabolism may differ between RAS-like and BRAF-like tumors and performed a study aimed at metabolomic profiling of TC. Methods: Based on Institutional Review Board approved protocol, we obtained fresh frozen human TC samples with corresponding normal tissue and performed targeted metabolomics including oxidative phosphorylation (OXPHOS), glycolysis and tricarboxylic acid (TCA) markers using mass spectrometry (MS). The Trusight 500 gene panel was used to determine molecular signature of tumors. Similarly, we performed targeted metabolomics of 4 BRAF-like and 2 RAS-like human TC cell lines. The data were analyzed using Ingenuity Pathway Analysis (Qiagen). We also analyzed mRNA expression of OXPHOS and glycolytic enzymes in 496 human TC tissue samples of The Cancer Genome Atlas (TCGA). The division into BRAF-like and RAS-like TC was based on well-established BRAF-RAS score (BRS) ranging from -1 to +1 for BRAF-like and RAS-like TC, respectively. Results: The study cohort consisted of 20 patients, 13 with BRAF-like and 7 with RAS-like tumors, aged 43±12 years old, 90% (18/20) females, with an average tumor size of 3±1.9 cm, multifocal growth in 55% (11/20) patients, central neck lymph node (LN) metastases in 55% (11/20), lateral neck LN metastases in 15% (3/20) patients and no distant metastases. The tumor size tended to be larger in RAS-like cohort (BRAF-like 2.27±1.28 vs RAS-like 4.34±2.52, p=0.08), and LN metastases more prevalent in BRAF-like cohort (BRAF-like 45% vs RAS-like 10%, p=0.08). Metabolomics data revealed that compared with normal thyroid, human TC tissue samples were characterized by an increased glucose metabolism (p<0.001), TCA (p<0.001) and gluconeogenesis (p<0.001). RAS-like tumors were characterized by a significantly higher TCA (p<0.001) and lower gluconeogenesis (p<0.001) as compared with BRAF-like tumors. Consistently, we found that TCA (p<0.001) and glucose metabolism (p<0.001) was higher in RAS-like vs BRAF-like cell lines. Transcriptomic profiling revealed that RAS-like TC was characterized by an increased expression of majority of tested OXPHOS enzymes compared to BRAF-like tumors, as evidenced by positive correlation with BRS, ranging from r = 0.28 to 0.54, p<0.001. Consistently, BRAF-like TC was associated with an upregulated mRNA expression of anaerobic glycolysis markers, with lactate dehydrogenase A, characterized by the highest negative correlation (r = -0.6, p<0.001) with BRS. Conclusion:RAS-like TC is more likely to utilize glucose through Krebs cycle, while BRAF-like tumors are more prone towards anaerobic glycolysis. Therapeutic strategies based on diverse metabolic profiles may provide a personalized approach to TC therapy. Presentation: 6/3/2024

Oncogenic mutations in the TP53 and PI-3 kinase/AKT pathway are independent predictors of survival for advanced thyroid cancer: Analysis from the Molecular Screening and Therapeutics (MoST) program

BackgroundThyroid cancers with mutations in the phosphatidylinositol-3 kinase/AKT pathway have a poorer prognosis. However, knowledge about the relationship between histology, mutation profile, and outcomes is still developing. This study assessed the prognostic value of genomic profiles for patients with advanced thyroid cancer who experienced progression on conventional treatment. MethodsPatients recruited to a national clinical oncology program for treatment-refractory locally advanced, recurrent or metastatic cancers were analyzed. Patients' archival tumor samples underwent comprehensive genomic profiling. Specific oncogenic mutations and the presence of cancer related pathways were correlated with overall survival. ResultsFrom 2018 to 2021, 4,955 patients were recruited, with 44 (0.9%) having a diagnosis of thyroid cancer with 4 medullary and the remaining follicular derived: 17 differentiated, 13 poorly differentiated, and 10 anaplastic thyroid cancers. Of the 40 follicular-derived thyroid cancer samples, 17 (42.5%) carried TP53 mutations, followed by 11 with BRAF V600E (27.5%), 9 with NRAS (22.5%), 9 with mutations in the phosphatidylinositol-3 kinase/AKT pathway (22.5%), and 7 with TERT promoter mutations (17.5%). Both TP53 and phosphatidylinositol-3 kinase/AKT pathway alterations were associated with reduced overall survival (hazard ratio, 5.19; 95% confidence interval, 1.59–16.70, P = .02 and hazard ratio, 10.12; 95% confidence interval, 1.61–63.76, P = .01). Cox regression showed histologic type anaplastic thyroid cancer (hazard ratio, 12.93, P = .004), poorly differentiated thyroid cancer (hazard ratio, 5.19, P = .039), and TP53 and/or phosphatidylinositol-3 kinase/AKT pathway mutations (hazard ratio, 4.73, P = .017) were independently associated with overall survival. ConclusionTP53 and/or phosphatidylinositol-3 kinase/AKT pathway mutations correlated with overall survival independently of histotype in patients with advanced thyroid cancer. Comprehensive genomic profiling has potential to inform prognosis, as well as identifying treatment targets for patients with advanced thyroid cancer.

Feasibility study of lncRNA DHRS4-AS1 sponge miR-222-3p in the diagnosis of thyroid cancer.

Thyroid cancer is a commonly occurring malignant tumour within the endocrine system, the incidence of which has been increasing steadily in our country. It has been the focus and direction of research in recent decades to continuously explore the diagnostic markers and molecular mechanisms of thyroid cancer and provide new possibilities for the healing of patients. In this study, lncRNA DHRS4-AS1 was identified as the research target, and the regulatory function of abnormal expression of DHRS4-AS1 on thyroid cancer was discussed. Thyroid cancer (116) and non-cancer normal (82) tissue samples were collected in this paper, and the expression of DHRS4-AS1 and miR-222-3p in tissues and cells were evaluated by RT-qPCR. CCK-8 and flow cytometry were used to detect cell survival status. The mechanism of DHRS4-AS1 sponge miR-222-3p was analysed by dual-luciferase reporter gene detection. In the present study, DHRS4-AS1 was down-regulated in both thyroid tissue and cell samples, while miR-222-3p expression was elevated. The ROC curve reflected the diagnostic value of DHRS4-AS1 in thyroid cancer [area under the curve (AUC) = 0.887, sensitivity = 76.7%, specificity = 95.1%]. DHRS4-AS1 regulates the development of thyroid cancer by targeting miR-222-3p. In addition, in vitro experiments demonstrated that overexpression of DHRS4-AS1 (pcDNA3.1-DHRS4-AS1) inhibited the proliferation of thyroid cancer cells and promoted cell apoptosis, while down-regulating the level of miR-222-3p. DHRS4-AS1 acts as a miR-222-3p sponge in thyroid cancer, and overexpression of DHRS4 AS1 down-regulates cell proliferation and promotes cell apoptosis. These findings demonstrate the potential of DHRS4-AS1 as a diagnostic factor for thyroid cancer.

High success rate of first proficiency testing for RET fusions and RET mutations in lung and thyroid cancer samples by various methods.

This study describes the external quality assessment (EQA) scheme for molecular testing of RET alterations in non-small cell lung cancer (NSCLC), medullary thyroid carcinomas (MTC), and non-MTC. The lead panel institute and Quality Assurance Initiative in Pathology (Qualitätssicherungs-Initiative Pathologie [QuIP] GmbH) selected formalin-fixed paraffin-embedded (FFPE) tissue from MTC for RET mutation testing by next-generation sequencing (NGS) methods and FFPE tissue from NSCLC and non-MTC for RET gene fusion testing using either in situ hybridisation (ISH) or NGS methods, forming 3 sub-schemes of the EQA scheme. Tissue material underwent an internal validation phase followed by an external testing phase. The internal validation phase served as a cross-validation step conducted by panel institutes. In the external testing phase, the number of participating institutes in the RET point mutation sub-scheme, RET fusion (ISH) sub-scheme, and RET fusion (NGS) sub-scheme was 32, 24, and 38, respectively. The reported success rates for external testing were 96.0%, 89.5%, and 93.5% for the RET point mutation, the ISH RET fusion, and the NGS RET fusion EQA sub-schemes, respectively. These findings confirm the reliability of the NGS method in detecting RET alterations and align with current screening recommendations. Overall, 31 institutes were certified for RET point mutation testing by NGS methods, 22 institutes were certified for RET fusion testing by ISH, and 36 institutes were certified for RET fusion testing by NGS methods. Results can be employed to inform real-world diagnostic decisions in Germany, Austria, and Switzerland.

In silico identification of differentially expressed microRNAs in thyroid cancer

BackgroundAbnormal expression of microRNAs is one of the crucial features contributing to the thyroid cancer (TC) progression. However, a comprehensive identification of the dysregulated microRNA profile and the associated molecular pathways that underlie the TC pathogenesis has not been completely provided. In the current study, bioinformatic analysis tools and microarray datasets were used to evaluate the biological roles of differentially expressed microRNAs and their targets in TC. MethodsGEO2R was used to identify differentially expressed microRNAs in TC samples. The mRNA targets of these microRNAs were predicted using different databases. DAVID and Reactome databases were used to perform gene ontology and pathway enrichment analyses of target genes. Then, the protein-protein interaction networks were constructed among them through the STRING database. MCODE was applied to screen hub genes. The prognostic values of hub genes were examined in TCGA THCA dataset using GEPIA2 platform. The relationship between hub genes and the ERBB2 protein was revealed using GeneMANIA. ResultsWe found a significant decrease in five microRNAs and a significant increase in five others in TC samples. Target genes of upregulated and downregulated microRNAs in TC were associated with ERBB2 signaling and ion exchanger pathways, respectively. CUX2 and DCUN1D4, the targets of upregulated microRNAs, were downregulated, whereas AP1S1, the target of downregulated microRNAs, were overexpressed in TCGA THCA samples. EZR and CUL5 were mediators for the interaction of ERBB2 with CUX2 or DCUN1D4, respectively. ConclusionWe suggest that CUX2/DCUN1D4 and AP1S1 may act as tumor suppressor and oncogene in TC onset and progression, respectively.

A novel exosome-related prognostic risk model for thyroid cancer.

The aim was to build an exosome-related gene (ERG) risk model for thyroid cancer (TC) patients. Note that, 510 TC samples from The Cancer Genome Atlas database and 121 ERGs from the ExoBCD database were obtained. Differential gene expression analysis was performed to get ERGs in TC (TERGs). Functional enrichment analyses including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted on the TERGs. Then we constructed a model based on LASSO Cox regression analysis. Kaplan-Meier survival analysis was applied and a Nomogram model was also built. The immune landscape was evaluated by CIBERSORT. Thirty-eight TERGs were identified and their functions were enriched on 591 GO terms and 30 KEGG pathways. We built a Risk Score model based on FGFR3, ADRA1B, and POSTN. Risk Scores were significantly higher in T4 than in other stages, meanwhile, it didn't significantly differ in genders and TNM N or M classifications. The nomogram model could reliably predict the overall survival of TC patients. The mutation rate of BRAF and expression of cytotoxic T-lymphocyte-associated protein 4 were significantly higher in the high-risk group than in the low-risk group. The risk score was significantly correlated to the immune landscape. We built a Risk Score model using FGFR3, ADRA1B, and POSTN which could reliably predict the prognosis of TC patients.

Unraveling the role of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer by multi-omics analyses

The role of the serine/glycine metabolic pathway (SGP) has recently been demonstrated in tumors; however, the pathological relevance of the SGP in thyroid cancer remains unexplored. Here, we perform metabolomic profiling of 17 tumor-normal pairs; bulk transcriptomics of 263 normal thyroid, 348 papillary, and 21 undifferentiated thyroid cancer samples; and single-cell transcriptomes from 15 cases, showing the impact of mitochondrial one-carbon metabolism in thyroid tumors. High expression of serine hydroxymethyltransferase-2 (SHMT2) and methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is associated with low thyroid differentiation scores and poor clinical features. A subpopulation of tumor cells with high mitochondrial one-carbon pathway activity is observed in the single-cell dataset. SHMT2 inhibition significantly compromises mitochondrial respiration and decreases cell proliferation and tumor size in vitro and in vivo. Collectively, our results highlight the importance of the mitochondrial one-carbon pathway in undifferentiated thyroid cancer and suggest that SHMT2 is a potent therapeutic target.

Impact of the quality of resected thyroid cancer tissue sample on next-generation sequencing testing.

Activating rearranged during transfection (RET) proto-oncogene alterations can be identified using next-generation sequencing (NGS) of tumor DNA/RNA. We assessed factors associated with NGS (Oncomine Dx Target Test [ODxTT]) success for resected thyroid cancer (TC) specimens, including sample age, processing conditions, and DNA/RNA quality. TC samples were from three Japanese hospitals, with sample age <1-<10 years, fixative 10%/15% neutralized buffered formalin (NBF), and fixation time ≤48 h/>48 h-≤72 h. NGS success rate was defined as the percentage of samples returning validated NGS results (RET fusion-positive/negative [RNA] or RET mutation-positive/negative [DNA], detected using ODxTT). DNA/RNA quality was assessed with indexes based on electrophoresis (DNA/RNA integrity number, DV200 ) and quantitative polymerase chain reaction (DNA/RNA integrity score [ddCq/ΔCq]). NGS success rate (N = 202) was 90%/93% (DNA/RNA) overall, 98%-100% (DNA and RNA) for samples <3 years old, and 91% (DNA and RNA) for samples ≥3-<5 years old fixed in 10% NBF for ≤48 h. Multivariate logistic regression analysis identified ddCq and ΔCq as significant predictors of DNA and RNA NGS success rates, respectively. Quality assessment of nucleic acid extracted from archival tissue samples is important for achieving high NGS success rates in clinical practice, especially for samples ≥3 years old.

Prognosis and therapy in thyroid cancer by gene signatures related to natural killer cells.

Natural killer (NK) cells are crucial to cancer development and prognosis. However, the role of NK cell-related genes in immunotherapy and the tumor immune microenvironment (TIME) is not well understood. This study aimed to develop reliable risk signatures associated with NK cell-related genes for predicting thyroid cancer (THCA). The single-cell RNA sequencing (scRNA-seq) data from seven THCA samples (GSE184362) and bulk-RNA-seq data of 502 THCA patients (TCGA-THCA) were included. The scRNA-seq data was analyzed using the "Seurat" R package to identify differentially expressed genes in NK cells. The clustering analysis was carried out using the R package "ConsensusClusterPlus". The gene set variation analysis (GSVA) algorithm was applied to assess the variations in biological pathways among subtypes. The ESTIMATE algorithm was utilized to calculate the scores for stromal, immune and estimate variables. In addition, we used the single sample Gene Set Enrichment Analysis and CIBERSORT algorithms to assess the degree to which immune cells and pathways related to immunity were enriched based on the meta-cohort. In the TCGA-THCA cohort, the "glmnet" R package was used for the gene selection, and LASSO Cox analysis was used to construct prognostic features. The "maftools" R package was used to examine the somatic mutation landscape of THCA in both low- and high-risk groups. One-hundred and eighty-five NK cell marker genes were screened, and nine genes were associated with the THCA prognosis. KLF2, OSTF1 and TAPBP were finally identified and constructed a risk signature with significant prognostic value. KLF2 and OSTF1 were protective genes, and TAPBP was a risk gene. Patients at high risk had a considerably lower overall survival compared with those at low risk. Mutations in the TCGA-THCA cohort were predominantly C > T. Increased tumor mutation burden (TMB) levels were linked to overall survival. The low-risk H-TMB+ group had a better prognosis, while the high-risk L-TMB+ group had the worst prognosis. Natural killer cell-related genes KLF2, OSTF1 and TAPBP were used to develop a novel prognostic risk signature, offering a new perspective on the prognosis and treatment of THCA.

Characterization of the Thyroid Cancer Genomic Landscape by Plasma-Based Circulating Tumor DNA Next-Generation Sequencing.

Background: The limited availability of targeted therapies in thyroid cancer (TC) has challenged conventional treatment algorithms and has established urgency for the identification of targetable genomic abnormalities. In addition to widely adopted tissue-based next-generation sequencing (NGS), plasma-based circulating tumor DNA (ctDNA) NGS is rapidly emerging as a genomic biomarker detection method and is steadily gaining utility across solid tumors. To date, plasma-based genomic alterations in TC have not been determined. Herein, we profile potential actionable mutations detected through ctDNA in patients with TC subtypes. Methods: A retrospective data analysis of the Guardant Health, Inc. database was performed using the commercially available Guardant360® plasma-NGS test on TC samples from adult patients collected between 2016 and 2021. The landscape of genomic alterations and blood tumor mutation burden (bTMB) were analyzed in patients with different types of TC: anaplastic TC (ATC), papillary TC (PTC), follicular TC (FTC), oncolytic carcinoma of the thyroid (OCA), poorly differentiated TC (PDTC), medullary TC (MTC), and TC not otherwise specified (TC NOS). Results: Of the 1094 patients included most of the patients n = 876 had TC NOS, and 20% had a specific diagnosis (92 ATC, 62 PTC, 14 FTC, 16 OCA, 2 PDTC, and 32 MTC patients). The median age was 65 (range 10-98) and 47.3% were male. 78.3% of patients had one or more genomic alteration detected by ctDNA NGS. TP53 (46.9%) was the most common mutation detected among all TC. BRAFV600E was detected in 27.2% of ATC, 35.7% of PTC, and in none of FTC. RAS was detected in 18.5% of ATC, 11.9% of PTC, and 62.5% of FTC. RET, ALK, and NTRK fusions were seen in 1.1%, 0.5%, and 0.2% of all TC, respectively. RET mutations were detected in 66.7% of MTC. bTMB analysis was performed on 159 patients. The mean bTMB was higher in ATC compared with other types of TC (p = 0.0011, 0.0557, and <0.0001, respectively). Conclusions: Plasma-based comprehensive NGS is a promising NGS method in TC; however, future validation of the clinical utility by analysis of paired tumor and plasma samples is needed.

Association of Cutibacterium acnes with human thyroid cancer.

The diverse subtypes of thyroid carcinoma have distinct clinical outcomes despite a comparable spectrum of underlying genetic alterations. Beyond genetic alterations, sparse efforts have been made to characterize the microbes associated with thyroid cancer. In this study, we examine the microbial profile of thyroid cancer. We sequenced the whole transcriptome of 70 thyroid cancers (40 papillary and 30 anaplastic). Using Infectious Pathogen Detector IPD 2.0, we analysed the relative abundance of 1060 microbes across 70 tumours from patients with thyroid cancer against 118 tumour samples from patients with breast, cervical, colorectal, and tongue cancer. Our analysis reveals a significant prevalence of Cutibacterium acnes in 58.6% thyroid cancer samples compared to other cancer types (p=0.00038). Immune cell fraction analysis between thyroid cancer samples with high and low Cutibacterium loads identify enrichment of immunosuppressive cells, including Tregs (p=0.015), and other anti-inflammatory cytokines in the tumour microenvironment, suggesting an immune evasion/immunosuppression milieu is associated with the infection. A higher burden of Cutibacterium acnes was also found to be associated with poor survival defining a distinct sub-group of thyroid cancer. Cutibacterium acnes is associated with immune suppression and poor prognosis in a subpopulation of thyroid cancer. This study may help design novel therapeutic measures involving appropriate antibiotics to manage the disease better.

THU484 Distinct Genetic Characteristics In Follicular Thyroid Cancer With Different Metastatic Potentials

Abstract Disclosure: S. Kwak: None. E. Lee: None. Y. Park: None. Although most follicular thyroid cancers (FTCs) show excellent prognosis, more than 5% accompany distant metastasis, often diagnosed unexpectedly in the advanced stage. Genetic or genomic markers have been shown to be useful as a prognostic tool in many cancers, but it is unclear whether genetic markers predicting the metastatic potential of FTCs exist. To investigate, 11 pairs of matched formalin fixed paraffin embedded tissue samples of primary (PTM1) and metastatic tumors (MT) from FTC patients with distant metastasis were analyzed by whole exome sequencing. For validation, 78 unmatched FTC samples including 49 well-differentiated tumors, 15 poorly-differentiated tumors and 14 anaplastic thyroid cancer (ATC) samples were analyzed by targeted sequencing. Among the 11 paired PTM1 and MT tumors, 10 showed concordant driver mutations (NRAS, HRAS, DICER1, EIF1AX), and the TERT promoter mutation was concordantly found in 6 pairs. However, the concordance rate of other somatic mutations in paired samples were found to be only 16.2%. Other mutational characteristics such as mutational signatures and CNVs also differed, suggesting the independent subclonal evolution of the paired MT from the PTM1 after metastasis. Interestingly, the frequency of mutations including driver, TERT promoter, and other mutations was lower in primary tumors without metastases (PTM0). Mutational characteristics of PTM0 also differed from those of PTM1 and MT, suggesting that the mutational or molecular characteristics of PTs at the time of diagnosis may distinguish metastatic potential. In a pooled analysis including published data of follicular patterned tumors, we found that metastatic or aggressive tumors were more likely to have the RAS mutation, which tended to be coupled with TERT, DICER1, EIF1AX, and TP53, gradually from minimally invasive FTC, widely invasive FTC to poorly differentiated thyroid cancer (PDTC) and ATC. Transcriptomic profiles from The Cancer Genome Atlas (TCGA) and our data (SNUH) showed RAS(+)/TERT(+) mutated tumors and RAS(+)/TERT(-) tumors had differences in several intracellular signal pathways. Therefore, when follicular neoplasm is diagnosed, NRAS and HRAS mutation tests followed by TERT promoter mutation tests may help diagnose FTCs with metastatic potential. Other genetic mutations could be candidates for a diagnostic or prognostic marker, and large-scale validation studies are warranted.This study was funded by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Science, ICT &amp; Future Planning (NRF-2019R1A2C2084332). Presentation: Thursday, June 15, 2023

Deltex E3 ubiquitin ligase 4 promotes thyroid cancer progression through stearoyl-CoA desaturase 1.

In this study, we aimed to explore the molecular role of Deltex E3 ubiquitin ligase 4 (DTX4) in thyroid cancer (TC) both in vitro and in vivo. The expression level of DTX4 in TC tissues was compared using The Cancer Genome Atlas (TCGA) database. We subsequently evaluated cell proliferation and migration in DTX4 knock down or DTX4 overexpression TC cell lines (TPC-1 and K1) by CCK-8, cell colony formation, and transwell assays. RNA sequencing and KEGG analysis were employed to identify potential genes that interact with DTX4. Our results showed that DTX4 was expressed at higher levels in both TC tissues and cells compared to normal controls. Knock down of DTX4 expression significantly inhibited TC cell progression in vitro. Furthermore, knockdown of endogenous DTX4 by shDTX4 markedly abrogated tumor growth, with significantly smaller tumor size and lower tumor weight in the shDTX4 group compared to the shCtrl group. Conversely, overexpression of DTX4 enhanced TC cell proliferation and migration. Through RNA sequencing, we identified 590 Differentially Expressed Genes (DEGs), with stearoyl-CoA desaturase 1 (SCD) ranking as the top gene. A positive correlation between DTX4 and SCD was observed in TC samples. Additionally, treatment with an SCD inhibitor, A939572, significantly rescued the enhanced growth effect induced by DTX4 overexpression. In conclusion, this study demonstrated that DTX4 promotes TC progression through SCD, indicating that the DTX4/SCD axis could be a promising target for TC therapy.

An investigation on the role of differentially expressed genes in thyroid cancer under the influence of hypoxia

Thyroid cancer is a common endocrine malignancy with a significant increase in its incidence in the past three decades. Even though research has significantly aided the management of the disease, the progression towards advanced forms of cancers remains indeterminate. In order to investigate the current challenges in thyroid cancer studies, the present work employed systematic and interactive transcriptomic data to construct plausible protein-protein interaction networks to reveal the putative transcriptional control mechanisms in cancer. The data from 4 different datasets consisting of normal samples vs thyroid cancer samples were chosen. Hypoxia being a significant hallmark of cancer was predicted to have a functional role in the progression of cancer. Consequently, prognostic pathways involved in cancer in response to hypoxia were predicted in the present study. The genes from the datasets were intersected with the hypoxia hallmark gene set to detect the significantly differentially expressed genes which were deregulated under the influence of hypoxia. These genes were analyzed by bioinformatic tools and a high correlation was found between 12 significant genes (PLAUR, BGN, SDC2, DUSP1, FOS, EGFR, CP, PPARGC1A, CITED2, RORA, HSPA5 and ACKR3) indicating a significant association between them. Of all the genes PLAUR was found to be novel and it was significantly upregulated under the influence of hypoxia. The hub genes and their role as predicted biomarkers were also determined by ROC curve analysis. This may assist in further research towards understanding role of hypoxia in Thyroid cancer.

Identification of Chimeric NTRK3 Genes in Papillary Thyroid Cancer Cells by Analyzing the Imbalance of the Expression of 5' and 3' mRNA Fragments.

The standard for detecting chimeric genes of neurotrophic receptor tyrosine kinases (NTRK) is next generation sequencing (NGS). However, this analysis is expensive and takes several days. As a rapid screening method for the detection of NTRK3-dependent papillary thyroid cancer, an analysis of the expression imbalance between 5' and 3' NTRK3 mRNA fragments was used (5'/3' RT-PCR). The reference method for detection of NTRK3 rearrangements was fluorescent in situ hybridization (FISH), and the most frequent rearrangements in papillary thyroid cancer were tested using reverse transcription PCR (RT-PCR). Using 5'/3' RT-PCR, 18 samples of papillary thyroid cancer carrying chimeric transcripts of NTRK3 mRNA were detected. The sensitivity of the developed technique was 88.9% and specificity was 99.3%. Thus, a fast and cost-effective method of screening samples of papillary thyroid cancer in paraffin blocks is proposed with acceptable sensitivity and specificity.

The RCAN1.4 Metastasis Suppressor Is Hypermethylated at Intron 1 in Thyroid Cancer.

Background: Regulator of calcineurin 1.4 (RCAN1.4) is a functionally downregulated metastasis progression suppressor (MPS) in thyroid cancer; however, the mechanisms for RCAN1.4 loss in thyroid cancer have not yet been reported. The RCAN1.4 promoter and gene contain several cytosine-guanine (CG)-rich regions, some of which are reported to be hypermethylated in nonthyroid tissues. We, therefore, hypothesized that RCAN1.4 downregulation in thyroid cancer was in part due to hypermethylation. Methods: Studies were performed in 5 thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers, and in 18 paired normal and thyroid cancer human thyroid cancer tissues. Basal RCAN1.4 messenger RNA (mRNA) and protein levels were assessed in all of the cell lines. Cell lines with lowest RCAN1.4 expression levels were treated with the DNA methyl transferase inhibitor, decitabine. Normal/tumor tissue pairs were analyzed for methylation of three CG-rich regions both by capture of methylated DNA by MBD2 protein and by methylation-specific polymerase chain reaction (MSPCR). Results: In all assessed cell lines, RCAN1.4 mRNA and protein levels increased after decitabine treatment. In silico analysis of the RCAN1.4 gene identified 3 CG-rich regions as possible methylation targets: 1 in the proximal promoter and 2 in intron 1. Hypermethylation of the intron 1 CG-rich regions was identified by both the capture method and MSPCR. In contrast, hypermethylation of the CG-rich region of the proximal promoter was not identified. Gene expression confirmed that hypermethylation in thyroid cancer samples in intron 1 of RCAN1.4 was associated with lower levels of RCAN1.4 mRNA. Finally, the cancer samples demonstrated increased NFE2L3 expression, a downstream marker of functional RCAN1.4 loss. Conclusions: The MPS gene, RCAN1.4, is downregulated in thyroid cancer cells and human thyroid cancer in part by hypermethylation of CG-rich regions in intron 1.

YTHDC2 Retards Cell Proliferation and Triggers Apoptosis in Papillary Thyroid Cancer by Regulating CYLD-Mediated Inactivation of Akt Signaling.

N6-Methyladenosine (m6A) mRNA methylation modification is regarded as an important mechanism involved in diverse physiological processes. YT521-B homology (YTH) domain family members are associated with the tumorigenesis of several cancers. However, the role of YTHDC2 in papillary thyroid cancer (PTC) progression remains unknown. Results showed that YTHDC1, YTHDF1, YTHDF2, and YTHDF3 showed no observable difference in thyroid cancer samples. YTHDC2 was significantly downregulated in thyroid cancer samples and cells. YTHDC2 inhibited cell proliferation in PTC cells. YTHDC2 elicited apoptosis in PTC cells, as demonstrated by the elevated expression of pro-apoptotic factors cl-caspase-3/caspase-3 and Bcl-2-associated (Bax), and the reduced anti-apoptotic B cell lymphoma-2 (Bcl-2) expression. There was a positive correlation between YTHDC2 and cylindromatosis (CYLD) expression based on GEPIA database. YTHDC2 increased CYLD expression in PTC cells. CYLD knockdown abolished the effects of YTHDC2 on PTC cell proliferation and apoptosis. Additionally, YTHDC2 inactivated the protein kinase B (Akt) pathway by increasing CYLD in PTC cells. Overall, YTHDC2 inhibited cell proliferation and induced apoptosis in PTC cells by regulating CYLD-mediated inactivation of Akt pathway.

Differences in Transcriptomic Profiles of Brain and Thyroid Tumors with NTRK Gene Rearrangement.

For tumors with chimeric NTRK genes, entrectinib and larotrectinib can be prescribed regardless of tumor localization. We compared changes in the transcriptional activity of genes in brain tumors (BT) and thyroid cancer (TC) with rearrangement (NTRK+) and without rearrangement (NTRK-) of the NTRK genes using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We revealed an increase in the transcription of the JUN gene in NTRK+ samples in comparison with NTRK- samples: by 1.6 times for BT (p=0.239) and by 2.5 times for TC (p=0.003). The transcription of eight HOX genes in NTRK+ BT samples was also increased (by 85-725 times, p<0.05) in comparison with NTRK-. In NTRK+ TC samples, the level of miR-31 and miR-542 was statistically significantly higher (by 3 and 2.5 times, respectively) than in NTRK-samples. For the NTRK+ BT samples, the levels of miR-10b, miR-182, and miR-21 more than 5-fold surpassed the corresponding values in NTRK-samples (p<0.05). These findings reflect differences in activation of gene transcription resulting from NTRK gene rearrangement in BT and TC.

Abstract 228: Recurrent mitochondrial mutations in thyroid cancer

Abstract Despite the importance of mitochondrial function on the bioenergetics and metabolism of the cell, understanding the functional impact of somatic mitochondrial mutations has been difficult. Additionally, small cohorts, as used in previous studies, lack the power to estimate the prevalence of specific recurrent mutations. We evaluated 177540 advanced cancer patients, across 84 different tumor types, who were sequenced as part of routine clinical care on the FoundationOne®CDx platform. Off-target sequencing reads were mapped to the mitochondrial genome (rCRS) and variant calling was performed. We had a median read depth of 18 and filtering by allele frequency (&amp;lt;= 95% and &amp;gt;= 5%) led to the identification almost 400,000 predicted somatic mitochondrial variants. Over 80% of patients had at least one mitochondrial variant detected, with an average of 2.2 mitochondrial variants detected per patient. We next assessed each recurrent mitochondrial short variant to look for associations with specific cancers. The alteration with the strongest enrichment was G3224A, a mutation in the mitochondrial leucine tRNA gene (MT-TL1), which had a prevalence of 3.6% (51/1426) in thyroid cancer samples, as compared to 0.05% (85/176114) in all other disease types (OR=76.8, P&amp;lt;2.2e-16). The G3244A mutation has been previously identified as occurring in thyroid cancer, specifically Hurthle cell carcinomas, as well as in the mitochondrial disease MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes). Thyroid cancer samples in our dataset with G3244A were found to be associated with thyroid follicular carcinomas (7/124, 5.6%) and Hurthle cell carcinomas (7/57, 12.2%). Additional recurrent MT-TL1 mutations were identified in the thyroid cancer cohort at positions 3242 (9/1426) and 3248 (5/1426). We also identified a mutation in the NADH dehydrogenase subunit MT-ND1. G3380A has been previously shown to occur in thyroid and colorectal cancer and results in a R25Q mutation that is thought to affect ND1 function. G3380A was identified in 26/1426 (1.8%) of thyroid cancer patients. The majority of G3380A variants occurred in thyroid anaplastic carcinomas 23/26 (88.5%). Our dataset has allowed us to identify and determine the prevalence of rare mitochondrial short variant alterations in cancer. Further research is needed to investigate associations of these alterations with other clinical features. Citation Format: Julia F. Hopkins, Zoe Fleischmann, Dexter X. Jin, Jonathan Keith Killian, Meagan Montesion. Recurrent mitochondrial mutations in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 228.