Analytical Accuracy of RET Fusion Detection by Break-Apart Fluorescence In Situ Hybridization.

CONTEXT.­: RET gene fusions are oncogenic drivers in nonsmall cell lung cancer and nonmedullary thyroid cancer. Selpercatinib (RETEVMO), a targeted inhibitor of RET, was approved by the US Food and Drug Administration for the treatment of RET fusion-positive nonsmall cell lung cancer and nonmedullary thyroid cancer emphasizing the need for rapid and accurate diagnosis of RET fusions. Fluorescence in situ hybridization (FISH) has been used to detect gene rearrangements, but its performance detecting RET rearrangements is understudied. OBJECTIVE.­: To validate and describe the performance of Abbott Molecular RET break-apart FISH probes for detecting RET rearrangements. DESIGN.­: A training set with RET fusion-positive (13) and RET fusion-negative nonsmall cell lung cancer and nonmedullary thyroid cancer samples (12) was used to establish criteria for FISH scoring. The scoring criteria was then applied to a larger validation set of samples (96). RESULTS.­: A cutoff of 19% or more positive nuclei by FISH was established in the training set and determined by the mean ±3 SD. The validation set was tested using Abbott Molecular RET break-apart FISH compared with sequencing. With this cutoff, a sensitivity of 86% (12 of 14) and specificity of 99% (81 of 82) was achieved. Bootstrapping showed sensitivity could be optimized by using a greater than 13% cutoff with indeterminate samples of 13% to 18% abnormal nuclei requiring confirmation by an orthogonal method. Using this 3-tier scoring system sensitivity increased to 100% (14 of 14) and specificity was 96% (79 of 82). CONCLUSIONS.­: Abbott Molecular break-apart FISH probes can be used to detect RET fusions. Laboratories can optimize cutoffs and/or testing algorithms to maximize sensitivity and specificity to ensure appropriate patients receive effective, timely therapy.

ABCG1 is required for pulmonary B-1 B cell and natural antibody homeostasis.

Many metabolic diseases, including atherosclerosis, type 2 diabetes, pulmonary alveolar proteinosis, and obesity, have a chronic inflammatory component involving both innate and adaptive immunity. Mice lacking the ATP-binding cassette transporter G1 (ABCG1) develop chronic inflammation in the lungs, which is associated with the lipid accumulation (cholesterol, cholesterol ester, and phospholipid) and cholesterol crystal deposition that are characteristic of atherosclerotic lesions and pulmonary alveolar proteinosis. In this article, we demonstrate that specific lipids, likely oxidized phospholipids and/or sterols, elicit a lung-specific immune response in Abcg1(-/-) mice. Loss of ABCG1 results in increased levels of specific oxysterols, phosphatidylcholines, and oxidized phospholipids, including 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine, in the lungs. Further, we identify a niche-specific increase in natural Ab (NAb)-secreting B-1 B cells in response to this lipid accumulation that is paralleled by increased titers of IgM, IgA, and IgG against oxidation-specific epitopes, such as those on oxidized low-density lipoprotein and malondialdehyde-modified low-density lipoprotein. Finally, we identify a cytokine/chemokine signature that is reflective of increased B cell activation, Ab secretion, and homing. Collectively, these data demonstrate that the accumulation of lipids in Abcg1(-/-) mice induces the specific expansion and localization of B-1 B cells, which secrete NAbs that may help to protect against the development of atherosclerosis. Indeed, despite chronic lipid accumulation and inflammation, hyperlipidemic mice lacking ABCG1 develop smaller atherosclerotic lesions compared with controls. These data also suggest that Abcg1(-/-) mice may represent a new model in which to study the protective functions of B-1 B cells/NAbs and suggest novel targets for pharmacologic intervention and treatment of disease.

Perilipin-5 is regulated by statins and controls triglyceride contents in the hepatocyte.

BACKGROUND & AIMS: Perilipin-5 (PLIN5) is a member of the perilipin family of lipid droplet (LD)-associated proteins. PLIN5 is expressed in oxidative tissues including the liver, and is critical during LD biogenesis. Studies showed that statins reduce hepatic triglyceride contents in some patients with non-alcoholic fatty liver disease and in rodent models of diet-induced hepatosteatosis. Whether statins alter triglyceride synthesis, storage, and/or utilization within the hepatocyte is unknown, though. Here we tested the hypothesis that statins alter the metabolism of LD in the hepatocyte during physiological conditions, such as fasting-induced steatosis. METHODS: Mice were gavaged with saline or atorvastatin, and the expression of LD-associated genes was determined in fed and fasted animals. The accumulation of triglycerides and LD was studied in mouse or human primary hepatocytes in response to statins, and following knock-down of SREBP2 or PLIN5. RESULTS: We show that statins decrease the levels of PLIN5, but not other LD-associated genes, in both mouse liver and mouse/human primary hepatocytes, which is paralleled by a significant reduction in both intracellular triglycerides and the number of LD. We identify an atypical negative sterol regulatory sequence in the proximal promoter of mouse/human PLIN5 that recruits the transcription factor SREBP2 and confers response to statins. Finally, we show that the statin-dependent reduction of hepatocyte triglyceride contents is mimicked by partial knock-down of PLIN5; conversely, ectopic overexpression of PLIN5 reverts the statin effect. CONCLUSIONS: PLIN5 is a physiological regulator of triglyceride metabolism in the liver, and likely contributes to the pleiotropic effects of statins.

Control of very low-density lipoprotein secretion by N-ethylmaleimide-sensitive factor and miR-33.

RATIONALE: Several reports suggest that antisense oligonucleotides against miR-33 might reduce cardiovascular risk in patients by accelerating the reverse cholesterol transport pathway. However, conflicting reports exist about the impact of anti-miR-33 therapy on the levels of very low-density lipoprotein-triglycerides (VLDL-TAG). OBJECTIVE: We test the hypothesis that miR-33 controls hepatic VLDL-TAG secretion. METHODS AND RESULTS: Using therapeutic silencing of miR-33 and adenoviral overexpression of miR-33, we show that miR-33 limits hepatic secretion of VLDL-TAG by targeting N-ethylmaleimide-sensitive factor (NSF), both in vivo and in primary hepatocytes. We identify conserved sequences in the 3'UTR of NSF as miR-33 responsive elements and show that Nsf is specifically recruited to the RNA-induced silencing complex following induction of miR-33. In pulse-chase experiments, either miR-33 overexpression or knock-down of Nsf lead to decreased secretion of apolipoproteins and TAG in primary hepatocytes, compared with control cells. Importantly, Nsf rescues miR-33-dependent reduced secretion. Finally, we show that overexpression of Nsf in vivo increases global hepatic secretion and raises plasma VLDL-TAG. CONCLUSIONS: Together, our data reveal key roles for the miR-33-NSF axis during hepatic secretion and suggest that caution should be taken with anti-miR-33-based therapies because they might raise proatherogenic VLDL-TAG levels.

Anti-miR-33 therapy does not alter the progression of atherosclerosis in low-density lipoprotein receptor-deficient mice.

OBJECTIVE: To determine the efficacy of long-term anti-miR-33 therapy on the progression of atherosclerosis in high-fat, high-cholesterol-fed Ldlr(-/-) mice. METHODS AND RESULTS: Ldlr(-/-) mice received saline, or control or anti-miR-33 oligonucleotides once a week for 14 weeks. The treatment was effective, as measured by reduced levels of hepatic miR-33 and increased hepatic expression of miR-33 targets. Analysis of plasma samples revealed an initial elevation in high-density lipoprotein cholesterol after 2 weeks of treatment that was not sustained by the end of the experiment. Additionally, we found a significant increase in circulating triglycerides in anti-miR-33-treated mice, compared with controls. Finally, examination of atheromata revealed no significant changes in the size or composition of lesions between the 3 groups. CONCLUSIONS: Prolonged silencing of miR-33 fails to maintain elevated plasma high-density lipoprotein cholesterol and does not prevent the progression of atherosclerosis in Ldlr(-/-) mice.

miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity.

Bile secretion is essential for whole body sterol homeostasis. Loss-of-function mutations in specific canalicular transporters in the hepatocyte disrupt bile flow and result in cholestasis. We show that two of these transporters, ABCB11 and ATP8B1, are functional targets of miR-33, a micro-RNA that is expressed from within an intron of SREBP-2. Consequently, manipulation of miR-33 levels in vivo with adenovirus or with antisense oligonucleotides results in changes in bile secretion and bile recovery from the gallbladder. Using radiolabelled cholesterol, we show that systemic silencing of miR-33 leads to increased sterols in bile and enhanced reverse cholesterol transport in vivo. Finally, we report that simvastatin causes, in a dose-dependent manner, profound hepatotoxicity and lethality in mice fed a lithogenic diet. These latter results are reminiscent of the recurrent cholestasis found in some patients prescribed statins. Importantly, pretreatment of mice with anti-miR-33 oligonucleotides rescues the hepatotoxic phenotype. Therefore, we conclude that miR-33 mediates some of the undesired, hepatotoxic effects of statins.

miR-33 links SREBP-2 induction to repression of sterol transporters.

The sterol regulatory element binding protein 2 (SREBP-2) and the liver X receptor (LXR) control antagonistic transcriptional programs that stimulate cellular cholesterol uptake and synthesis, and cholesterol efflux, respectively. The clinical importance of SREBP-2 is revealed in patients with hypercholesterolemia treated with statins, which reduce low-density lipoprotein (LDL) cholesterol levels by increasing hepatic expression of SREBP-2 and its target, the LDL receptor. Here we show that miR-33 is encoded within SREBP-2 and that both mRNAs are coexpressed. We also identify sequences in the 3' UTR of ABCA1 and ABCG1, sterol transporter genes both previously shown to be regulated by LXR, as targets for miR-33-mediated silencing. Our data show that LXR-dependent cholesterol efflux to both ApoAI and serum is ameliorated by miR-33 overexpression and, conversely, stimulated by miR-33 silencing. Finally, we show that ABCA1 mRNA and protein and plasma HDL levels decline after hepatic overexpression of miR-33, whereas they increase after hepatic miR-33 silencing. These results suggest novel ways to manage hypercholesterolemic patients.