Targeting Tumor Hypoxia Inhibits Aggressive Phenotype of Dedifferentiated Thyroid Cancer.

CONTEXT: Hypoxia is commonly observed in multiple aggressive cancers. Its role remains unclear in the biology and therapy of dedifferentiated thyroid cancer (DDTC). OBJECTIVE: We aimed to elucidate hypoxia's roles in DDTC tumor biology. METHODS: We discovered and confirmed hypoxia's correlation with dedifferentiation status, poor prognoses, and immune checkpoints in thyroid cancer using transcriptome data from our center and Gene Expression Omnibus (GEO) database. Then, the effect of targeting hypoxia was investigated via treating anaplastic thyroid cancer (ATC) cells with acriflavine (ACF) in vitro and in vivo, and hypoxia was analyzed for its association with response to immunotherapy in patients. RESULTS: Hypoxia score was positively associated with dedifferentiation status, and high hypoxia score significantly correlated with reduced overall survival, TP53 mutation, and elevated expression of immunosuppression-related markers in DDTC. ACF and siRNA targeting HIF-1α significantly suppressed growth and proliferation of thyroid cancer cells in vitro and in vivo, and reduced c-MYC and PDL1 expression in ATC. HIF-1α showed a positive correlation with PDL1 expression in DDTC. Integrated analyses of phosphoproteome and RNA sequencing data revealed that ACF's target was connected with differentiation genes and immune checkpoints via tumor-related kinases in ATC. Furthermore, hypoxia score was associated with immunotherapeutic response in some cancer types. CONCLUSION: Hypoxia score serves as a significant indicator for dedifferentiation status, prognoses, and immunotherapeutic response predicted by Tumor Immune Dysfunction and Exclusion in DDTC patients. Targeting hypoxia by ACF is useful to alleviate aggressive phenotype of ATC in a preclinical model of DDTC.

Identification of lipid metabolism-related genes as prognostic indicators in papillary thyroid cancer.

Lipid metabolism plays important roles not only in the structural basis and energy supply of healthy cells but also in the oncogenesis and progression of cancers. In this study, we investigated the prognostic value of lipid metabolism-related genes in papillary thyroid cancer (PTC). The recurrence predictive gene signature was developed and internally and externally validated based on PTC datasets including The Cancer Genome Atlas (TCGA) and GSE33630 datasets. Univariate, LASSO, and multivariate Cox regression analysis were applied to assess prognostic genes and build the prognostic gene signature. The expression profiles of prognostic genes were further determined by immunohistochemistry of tissue microarray using in-house cohorts, which enrolled 97 patients. Kaplan-Meier curve, time-dependent receiver operating characteristic curve, nomogram, and decision curve analyses were used to assess the performance of the gene signature. We identified four recurrence-related genes, PDZK1IP1, TMC3, LRP2 and KCNJ13, and established a four-gene signature recurrence risk model. The expression profiles of the four genes in the TCGA and in-house cohort indicated that stage T1/T2 PTC and locally advanced PTC exhibit notable associations not only with clinicopathological parameters but also with recurrence. Calibration analysis plots indicate the excellent predictive performance of the prognostic nomogram constructed based on the gene signature. Single-sample gene set enrichment analysis showed that high-risk cases exhibit changes in several important tumorigenesis-related pathways, such as the intestinal immune network and the p53 and Hedgehog signaling pathways. Our results indicate that lipid metabolism-related gene profiling represents a potential marker for prognosis and treatment decisions for PTC patients.

Application of preoperative computed tomographic lymphography for precise sentinel lymph node biopsy in breast cancer patients.

BACKGROUND: In light of the extensive application of sentinel lymph node biopsy (SLNB) in clinically node-negative breast cancer patients and the recently investigated failure of SLNB after lumpectomy, it has become important to explore methods for preoperative mapping of sentinel lymph nodes (SLNs) and their lymphatics to direct precise SLNB and improve the identification rate of SLNs. METHODS: Twenty-seven patients with suspected breast cancer based on the results of the clinical examination and imaging were enrolled in the study. Computed tomographic lymphography (CTLG) followed by CT three-dimensional reconstruction was performed to determine the localization of SLNs and lymphatics on the body surface preoperatively. Intraoperatively combined staining with methylene blue and indocyanine green was used to evaluate the accuracy and feasibility of CTLG. RESULTS: SLNs and lymphatics from the breast were identified using CTLG in all patients, and preoperative SLNs and lymphatics localization on the body surface showed a significant role in the selection of operative incision and injection points. The accuracy rate of SLN and lymphatic detection by CTLG was 92.6% compared with intraoperatively combined staining. Moreover, preoperative CTLG performed well in SLN number detection, and the accuracy rate was 95.2%. CONCLUSION: We evaluate the procedure and application of preoperative CTLG in the superficial localization of SLNs and lymphatics, which may lead to a decreased incidence of cutting off the lymphatics of SLNs and consequently more rapid and accurate SLN detection. This method promotes personalized SLN mapping, providing detailed information about the number and anatomical location of SLNs and lymphatics for adequate surgical planning for breast cancer patients.

Cancer-Associated Fibroblasts Positively Correlate with Dedifferentiation and Aggressiveness of Thyroid Cancer.

BACKGROUND AND OBJECTIVES: Targeting cancer-associated fibroblast (CAF) is being explored as an approach to improve cancer therapies. The roles of CAF remain unclarified in malignant transformation of papillary thyroid cancer (PTC) into dedifferentiated thyroid cancer (DDTC). This study aimed to investigate correlations of CAF with dedifferentiation and clinicopathological characteristics of thyroid cancer. MATERIALS AND METHODS: We applied three different mRNA-based CAF gene signatures to quantify CAF in our cohort, the Gene Expression Omnibus (GEO) cohort and The Cancer Genome Atlas (TCGA) cohort, and analyzed expression of α-SMA by immunohistochemistry in thyroid cancer. The CAF score was analyzed for its associations with clinicopathological characteristics, genetic mutations, tumor-associated signaling pathways and immune landscape. RESULTS: The CAF score increased significantly in DDTCs compared with normal thyroid tissues and PTCs, and the α-SMA-positive CAFs were found enriched in DDTCs. The high CAF score showed a significant correlation with the anaplastic phenotype in DDTC and low thyroid differentiation score in PTC. Patients with a high CAF score remarkably increased the risk of aggressive outcomes in both DDTC and PTC. Furthermore, the CAF score was positively correlated with genetic mutations, oncogenic signaling pathways, the immune score and increased expression of tumor microenvironment (TME) target markers. CONCLUSION: Our findings suggest CAFs positively correlate with dedifferentiation and aggressive outcomes of thyroid cancer, and targeting CAFs as a therapeutic approach may benefit DDTC patients.

Immune Co-inhibitory Receptors PD-1, CTLA-4, TIM-3, LAG-3, and TIGIT in Medullary Thyroid Cancers: A Large Cohort Study.

CONTEXT: Programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), and T-cell immunoglobulin and ITIM domain (TIGIT) are considered major immune co-inhibitory receptors (CIRs) and the most promising immunotherapeutic targets in cancer treatment, but they are largely unexplored in medullary thyroid carcinoma (MTC). OBJECTIVE: We aimed to provide the first evidence regarding the expression profiles and clinical significance of CIRs in a large cohort of MTC patients. DESIGN AND PATIENTS: In total, 200 MTC patients who received initial surgery in our hospital were included. Immunohistochemistry was performed to evaluate CIR expressions in tissue microarrays (TMAs). Combined with the results of our previous programmed cell death ligand-1 (PD-L1) study, clinicopathologic and prognostic correlations of these proteins were retrospectively analyzed. RESULTS: TIM-3, PD-1, CTLA-4, LAG-3, and TIGIT positivity was detected in 96 (48.0%), 27 (13.5%), 25 (12.5%), 6 (3.0%), and 6 (3.0%) patients, respectively, in whom TIM-3, PD-1, and CTLA-4 expressions were positively correlated. Log-rank tests and multivariate Cox analyses both indicated that TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression were associated with worse structural recurrence-free survival. In addition, among 20 patients who developed advanced disease during follow-up, 12 (60%) showed TIM-3 positivity, among whom 6 cases also had concurrent moderate to strong PD-1, PD-L1, or CTLA-4 expression. CONCLUSIONS: Using the currently largest TMA cohort of this rare cancer, we delineated the CIR expression profiles in MTC, and identified TIM-3, CTLA-4 expression, and PD-1/PD-L1 coexpression as promising biomarkers for tumor recurrence. Furthermore, a subset of advanced MTCs are probably immunogenic, for which single or combined immunotherapy including TIM-3, PD-1, PD-L1, or CTLA-4 blockade may be potential therapeutic approaches in the future.

A Novel Three-lncRNA Signature Predicts the Overall Survival of HNSCC Patients.

BACKGROUND: A number of long non-coding RNAs (lncRNAs) have been found to be involved in tumor progression and associated with disease prognoses in various types of cancer. Our study identified a novel three-lncRNA signature to predict survival of head and neck squamous cell caner (HNSCC) patients. METHODS: We utilized The Cancer Genome Atlas (TCGA) cohort to screen out overall survival (OS)-associated lncRNAs in HNSCC and further developed a model to identify a lncRNA signature for evaluating disease status and prognosis. The lncRNA signature was then validated in HNSCC patients from our Fudan University Shanghai Cancer Center (FUSCC) cohort. RESULTS: LINC02434, AL139327.2, and AC126175.1 were identified by multivariable Cox regression analyses of independent risk factors for deceased status. We built a risk score model based on the three-lncRNA signature using coefficient of multivariable Cox regression and expression value of the three lncRNAs. The high-risk signature score was significantly associated with decreased OS in both the TCGA cohort and the FUSCC cohort. The high-risk group had worse overall survival than the low-risk group in TCGA cohort. To further validate the robustness of three-lncRNA signature risk score model developed in the TCGA dataset, the performance of risk score also evaluated in our institute FUSCC cohort. Additionally, the signature score showed a positive correlation with aggressive outcomes of HNSCC, such as III/IV stage, TP53 mutation, and PI3KCA mutation. The gene set enrichment analysis indicates that the risk score is associated with cancer metastasis-related pathways. Several cancer-related pathways, such as epithelial mesenchymal transition, TNFα signaling via NF-κB, MYC targets, and angiogenesis. CONCLUSIONS: The three-lncRNA signature could provide a novel prediction insight into the prognosis of HNSCC patients. The three-lncRNA signature was identified as a predictor of poor prognoses in HNSCC, which may serve as a potential therapeutic target.

Genomic and Transcriptomic Characterization of Sporadic Medullary Thyroid Carcinoma.

Background: Sporadic medullary thyroid carcinoma (MTC) is a relatively uncommon neuroendocrine malignancy and the molecular tumorigenesis of its sporadic type (sMTC) is only partially understood. In this study, we performed a study focusing on the genomic and transcriptomic characterization of sMTC. Methods: Twenty-nine sMTC patients were included. Whole-exome sequencing (WES) was carried out in 18 patients, including both tumor samples and matched noncancerous tissues. Whole transcriptome sequencing (RNA-Seq) was performed in all 29 tumors. WES, RNA-Seq, and copy number alteration (CNA) data were analyzed. A Cell Counting Kit-8 (CCK-8) assay was used to evaluate cell proliferation. Results: Among the somatic mutations, RET was the only recurrently cancer-related mutated gene (5/18, 27.8%). In the germline, FAT1 and FAT4, two members of the FAT gene family, were identified as the two most common mutated genes. CNA analysis found that FAT1 and FAT4, both located on chromosome 4q, were also two of the genes most commonly affected by somatic chromosomal deletions (4/18, 22.2%). Using TT and MZ-CRC-1 cell lines, the CCK-8 assay showed that FAT1 and FAT4 knockdown could promote MTC cell proliferation. Based on the gene expression profile, patients were clustered into two molecular subtypes: the mesenchymal-like subtype is characterized by epithelial-mesenchymal transition, while the proliferative-like subtype is associated with enrichment of cell cycle pathways. Most events of structural recurrence (80%) occurred in the proliferative-like subtype. Conclusion: In addition to RET, these findings demonstrate that FAT1/FAT4 genomic alterations appear to be frequent in sMTC. Two molecular subtypes of sMTC with distinct biological behavior could be identified. However, these results need to be validated by larger samples and more comprehensive experiments in the future, especially for the frequency and function of FAT1/FAT4 germline variants.

The association between tumor's location and cervical lymph nodes metastasis in papillary thyroid cancer.

BACKGROUND: Papillary thyroid cancer (PTC) has a strong propensity to metastasize to the cervical lymph nodes. Little was known currently about whether tumor's location would influence the risk of lymph node metastasis in PTC. METHODS: The study enrolled PTC patients who underwent primary surgical therapy in our center for small unifocal tumor. The tumor's location was evaluated by ultrasound in three axes, three planes and 3D space. Logistic univariate and multivariate analysis were applied to explore the association between tumors' location and the risk of lymph node metastasis in PTC. Different localization methods of thyroid tumors were evaluated using ROC curve. RESULTS: Totally 1,266 PTC patients were enrolled in this study. Univariate and multivariate analyses showed that gender, age, tumor size and tumor's location (in longitudinal axis, longitudinal sagittal plane, longitudinal coronal plane, sagittal coronal plane and 3D space) was associated with central lymph node dissection (CLND); gender, tumor size and tumor's location (in longitudinal axis, coronal axis, longitudinal sagittal plane, longitudinal coronal plane, sagittal coronal plane and 3D space) was related with lateral lymph node dissection (LLND) (P<0.05). In the ROC curve analysis, the 3D location showed the highest predictive value of lymph node metastasis (C-statistics: 0.724 for CLNM; 0.763 for LLNM). The middle posterior lateral (OR=2.575, P=0.028), inferior anterior central (OR=2.829, P=0.016), inferior posterior lateral (OR=2.759, P=0.039) and isthmus tumors (OR=4.526, P=0.001) were at a higher risk of CLNM, and the middle anterior central tumors (OR=0.102, P=0.015) were related with lower risk of LLNM. CONCLUSIONS: Stereotactic localization showed the highest predictive value of lymph node metastasis. The middle posterior lateral, inferior anterior central, inferior posterior lateral and isthmus tumors were at a higher risk of CLNM when compared to other locations. For such patients, careful preoperative evaluation of nodal status should be done.

Metabolic reprogramming and its clinical application in thyroid cancer.

Warburg found that tumor cells exhibit high-level glycolysis, even under aerobic condition, which is known as the 'Warburg effect'. As systemic changes in the entire metabolic network are gradually revealed, it is recognized that metabolic reprogramming has gone far beyond the imagination of Warburg. Metabolic reprogramming involves an active change in cancer cells to adapt to their biological characteristics. Thyroid cancer is a common endocrine malignant tumor whose metabolic characteristics have been studied in recent years. Some drugs targeting tumor metabolism are under clinical trial. This article reviews the metabolic changes and mechanisms in thyroid cancer, aiming to find metabolic-related molecules that could be potential markers to predict prognosis and metabolic pathways, or could serve as therapeutic targets. Our review indicates that knowledge in metabolic alteration has potential contributions in the diagnosis, treatment and prognostic evaluation of thyroid cancer, but further studies are needed for verification as well.

The application of selective neck dissection while preserving the cutaneous branches of cervical plexus in the surgical treatment of differentiated thyroid cancer-experiences from thousands of cases.

Differentiated thyroid cancer (DTC) is associated with the highest propensity for lymph node metastases, given the significant morbidity associated with sacrificing the spinal accessory nerve, surgeons increasingly looked to minimizing functional deficits while maintaining oncologic outcome. We have detailed the technique of a selective neck dissection with more attention to preserving the cervical sensory nerves since 1999 in Fudan University Shanghai Cancer Center. We found that the radical dissection with preservation of the cutaneous branches including the great auricular nerve, the less occipital nerve and the supraclavicular nerve can maximally decrease the complications of paresthesia and dysesthesia postoperatively in the lower neck, the shoulders and the area around the ear in DTC cases when indications were allowed. As long as the principles of cancer surgery are strictly followed, our approach guarantees radical tumor removal and exhibit more functional preservation.

Relationship between Upper Extremity Lymphatic Drainage and Sentinel Lymph Nodes in Patients with Breast Cancer.

PURPOSE: The purpose of this study was to identify the relationship between upper extremity lymphatics and sentinel lymph nodes (SLNs) in breast cancer patients. METHODS: Forty-four patients who underwent axillary reverse mapping (ARM) during axillary lymph node dissection (ALND) with SNL biopsy (SLNB) between February 2017 and October 2017 were investigated. ARM was performed using indocyanine green (ICG) to locate the upper extremity lymphatics; methylene blue dye was injected intradermally for SLN mapping. RESULTS: ARM nodes were found in the ALND fields of all examined patients. The rate of identification of upper extremity lymphatics within the SLNB field was 65.9% (29 of 44). The ARM nodes were involved in metastases arising from primary breast tumors in 7 of the patients (15.9%), while no metastases were detected in pathologic axillary lymph node-negative patients. Lymphatics from the upper extremity drained into the SLNs in 5 of the 44 patients (11.4%); their ARM-detected nodes were found to be in close proximity to the SLNs. CONCLUSIONS: The ARM nodes and SLNs are closely related and share lymphatic drainage routes. The ARM procedure using fluorescence imaging is both feasible and, in patients who are SLN negative, oncologically safe. ARM using ICG is therefore effective for identifying and preserving upper extremity lymphatics, and SLNB combined with ARM appears to be a promising surgical refinement for preventing upper extremity lymphoedema. CLINICAL TRIAL REGISTRATION: This trial is registered with ClinicalTrial.gov: NCT02651142.

Effects of tumor size on prognosis in differentiated thyroid carcinoma smaller than 2 cm.

Benefits of subdividing small-differentiated thyroid carcinoma (sDTC) by tumor size are controversial. We conducted a meta-analysis to investigate whether tumor size is associated with prognosis of sDTC. PubMed and Web of Science databases were searched from their inception to September 2018. The identified studies according to the inclusion/exclusion criteria were analyzed using fixed/random-effects models. Data were calculated and results of the meta-analysis were expressed as odd ratio (OR). sDTC was classified as S1 (≤1 cm) and S2 (>1 cm and ≤2 cm). A systematic analysis was performed to compare the difference of recurrence, survival and clinicopathological factors between the two subgroups of sDTC (S1 vs. S2). A total of 21 studies published between 2004 and 2017 enrolling 219,291 patients were included. Findings showed that, S2 was associated with higher recurrence risk compared with S1 (OR=1.575, 95% CI=1.428-1.738; P<0.05). There was no statistical difference in survival between S1 and S2, but significant statistical heterogeneity (OR=1.160, 95% CI=0.810-1.662; P=0.448; I2=75.8%). Meta-regression analysis revealed publication year potentially caused the heterogeneity (P<0.05). Comparison of small papillary thyroid carcinoma alone agreed with the results of sDTC. T1b increased the risk of recurrence (OR=1.520; 95% CI=1.072-2.155; P<0.05) and death (OR=1.504; 95% CI 1.353-1.672; P<0.05) compared with T1a. S2 associated with extrathyroidal extension (OR=2.575; 95% CI=1.603-4.135; P<0.05), bilaterality (OR=2.278; 95% CI=1.905-2.723; P<0.05), vascular invasion (OR=4.494; 95% CI=2.812-7.183; P<0.05) and lymph node metastases (OR=1.12; 95% CI=1.10-1.14; P<0.05). Our analysis suggested it is necessary to subdivide sDTC into S1 and S2 owing to their different effects on prognosis, especially recurrence.

Allosteric modulation of the catalytic VYD loop in Slingshot by its N-terminal domain underlies both Slingshot auto-inhibition and activation.

Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp361 in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu63, and the exposure of the loop between α-helix-2 and ß-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding in vitro and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET-based Slingshot2 biosensor whose readout was highly correlated with the in vivo phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity.

The second-sphere residue T263 is important for the function and catalytic activity of PTP1B via interaction with the WPD-loop.

Protein tyrosine phosphatases have diverse substrate specificities and intrinsic activities that lay the foundations for the fine-tuning of a phosphorylation network to precisely regulate cellular signal transduction. All classical PTPs share common catalytic mechanisms, and the important catalytic residues in the first sphere of their active sites have been well characterized. However, little attention has been paid to the second-sphere residues that are potentially important in defining the intrinsic activity and substrate specificity of PTPs. Here, we find that a conserved second-sphere residue, Thr263, located in the surface Q-loop is important for both the function and activity of PTPs. Using PTP1B as a study model, we found that mutations of Thr263 impaired the negative regulation role of PTP1B in insulin signaling. A detailed mechanistic study utilizing steady-state kinetics, Brønsted analysis and pH dependence in the presence of pNPP or phosphopeptide substrates revealed that Thr263 is required for the stabilization of the leaving group during catalysis. Further crystallographic studies and structural comparison revealed that Thr263 regulates the general acid function through modulation of the WPD-loop by the T263:F182/Y/H interaction pair, which is conserved in 26 out of 32 classical PTPs. In addition, the hydrophobic interaction between Thr263 and Arg1159 of the insulin receptor contributes to the substrate specificity of PTP1B. Taken together, our findings demonstrate the general role of the second-sphere residue Thr263 in PTP catalysis. Our findings suggest that the second sphere residues of PTP active site may play important roles in PTP-mediated function in both normal and diseased states.