Analytical performance of the ThyroSeq v3 genomic classifier for cancer diagnosis in thyroid nodules.

BACKGROUND: Molecular tests have clinical utility for thyroid nodules with indeterminate fine-needle aspiration (FNA) cytology, although their performance requires further improvement. This study evaluated the analytical performance of the newly created ThyroSeq v3 test. METHODS: ThyroSeq v3 is a DNA- and RNA-based next-generation sequencing assay that analyzes 112 genes for a variety of genetic alterations, including point mutations, insertions/deletions, gene fusions, copy number alterations, and abnormal gene expression, and it uses a genomic classifier (GC) to separate malignant lesions from benign lesions. It was validated in 238 tissue samples and 175 FNA samples with known surgical follow-up. Analytical performance studies were conducted. RESULTS: In the training tissue set of samples, ThyroSeq GC detected more than 100 genetic alterations, including BRAF, RAS, TERT, and DICER1 mutations, NTRK1/3, BRAF, and RET fusions, 22q loss, and gene expression alterations. GC cutoffs were established to distinguish cancer from benign nodules with 93.9% sensitivity, 89.4% specificity, and 92.1% accuracy. This correctly classified most papillary, follicular, and Hurthle cell lesions, medullary thyroid carcinomas, and parathyroid lesions. In the FNA validation set, the GC sensitivity was 98.0%, the specificity was 81.8%, and the accuracy was 90.9%. Analytical accuracy studies demonstrated a minimal required nucleic acid input of 2.5 ng, a 12% minimal acceptable tumor content, and reproducible test results under variable stress conditions. CONCLUSIONS: The ThyroSeq v3 GC analyzes 5 different classes of molecular alterations and provides high accuracy for detecting all common types of thyroid cancer and parathyroid lesions. The analytical sensitivity, specificity, and robustness of the test have been successfully validated and indicate its suitability for clinical use. Cancer 2018;124:1682-90. © 2018 American Cancer Society.

ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion.

ARTD1 (PARP1) is a key enzyme involved in DNA repair through the synthesis of poly(ADP-ribose) (PAR) in response to strand breaks, and it plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD(+) depletion and ATP loss; however, the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we compared the effects of ARTD1 activation and direct NAD(+) depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD(+) depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics-based PAR interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing insight into the importance of nucleus-to-mitochondria communication via ARTD1 activation.

Mitochondrial respiration--an important therapeutic target in melanoma.

The importance of mitochondria as oxygen sensors as well as producers of ATP and reactive oxygen species (ROS) has recently become a focal point of cancer research. However, in the case of melanoma, little information is available to what extent cellular bioenergetics processes contribute to the progression of the disease and related to it, whether oxidative phosphorylation (OXPHOS) has a prominent role in advanced melanoma. In this study we demonstrate that compared to melanocytes, metastatic melanoma cells have elevated levels of OXPHOS. Furthermore, treating metastatic melanoma cells with the drug, Elesclomol, which induces cancer cell apoptosis through oxidative stress, we document by way of stable isotope labeling with amino acids in cell culture (SILAC) that proteins participating in OXPHOS are downregulated. We also provide evidence that melanoma cells with high levels of glycolysis are more resistant to Elesclomol. We further show that Elesclomol upregulates hypoxia inducible factor 1-α (HIF-1α), and that prolonged exposure of melanoma cells to this drug leads to selection of melanoma cells with high levels of glycolysis. Taken together, our findings suggest that molecular targeting of OXPHOS may have efficacy for advanced melanoma.