Analysis of receptor tyrosine kinase ROS1-positive tumors in non-small cell lung cancer: identification of a FIG-ROS1 fusion.

PURPOSE: To deepen our understanding of mutant ROS1 expression, localization, and frequency in non-small cell lung cancer (NSCLC), we developed a highly specific and sensitive immunohistochemistry (IHC)-based assay that is useful for the detection of wild-type and mutant ROS1. EXPERIMENTAL DESIGN: We analyzed 556 tumors with the ROS1 D4D6 rabbit monoclonal antibody IHC assay to assess ROS1 expression levels and localization. A subset of tumors was analyzed by FISH to determine the percentage of these tumors harboring ROS1 translocations. Using specific and sensitive IHC assays, we analyzed the expression of anaplastic lymphoma kinase (ALK), EGFR L858R, and EGFR E746-A750del mutations in a subset of lung tumors, including those expressing ROS1. RESULTS: In our NSCLC cohort of Chinese patients, we identified 9 (1.6%) tumors expressing ROS1 and 22 (4.0%) tumors expressing ALK. FISH identified tumors with ALK or ROS1 rearrangements, and IHC alone was capable of detecting all cases with ALK and ROS1 rearrangements. ROS1 fusion partners were determined by reverse transcriptase PCR identifying CD74-ROS1, SLC34A2-ROS1, and FIG-ROS1 fusions. Some of the ALK and ROS1 rearranged tumors may also harbor coexisting EGFR mutations. CONCLUSIONS: NSCLC tumors with ROS1 rearrangements are uncommon in the Chinese population and represent a distinct entity of carcinomas. The ROS1 IHC assay described here is a valuable tool for identifying patients expressing mutant ROS1 and could be routinely applied in clinical practice to detect lung cancers that may be responsive to targeted therapies.

Recovery from liver disease in a Niemann-Pick type C mouse model.

Loss of function of Niemann-Pick C1 (NPC1) leads to lysosomal free cholesterol storage, resulting in the neurodegenerative disease Niemann-Pick disease type C (NPC). Significant numbers of patients with NPC also suffer from liver disease. Currently, no treatments exist that alter patient outcome, and it is unknown if recovery from tissue damage can occur even if a treatment were found. Our laboratory developed a strategy to test whether mice can recover from NPC liver disease. We used antisense oligonucleotides to knock down hepatic expression of NPC1 in BALB/C mice for either 9 or 15 weeks. This recapitulated liver disease with hepatomegaly, cell death, and fibrosis. Then, antisense oligonucleotide treatment was halted for an additional 4, 9, or 15 weeks. We report that significant liver recovery occurred even when NPC1 protein expression only partially returned to normal. Several pathological phenotypes were alleviated, including hepatomegaly, cholesterol storage, and liver cell death. Histological examination revealed that foamy cell accumulation was relieved; however, liver fibrosis increased. Additionally, resolution of cholesterol storage and liver cell death took longer in mice with long-term knockdown. Finally, we found that transcription of cholesterol homeostatic genes was significantly disrupted during the recovery phase after long-term knockdown.

TNF-{alpha} plays a role in hepatocyte apoptosis in Niemann-Pick type C liver disease.

Niemann-Pick type C (NPC) is a fatal autosomal recessive lysosomal storage disease clinically characterized by neurodegeneration and liver disease. Heterogeneous mutations in the NPC1 and NPC2 genes cause impaired egress of free cholesterol from lysosomes, leading to accumulation of cholesterol and glycosphingolipids. Key features of NPC liver disease include hepatic apoptosis, inflammation, and fibrosis. It is unclear what signaling events regulate these disease processes in NPC. We hypothesize that tumor necrosis factor alpha (TNF-alpha), which is involved in both proinflammatory and apoptotic signaling cascades, is a key mediator of inflammation, apoptosis, and fibrosis in NPC liver disease. In this study, we evaluated the role of TNF-alpha signaling in NPC liver disease by utilizing NPC1-specific antisense oligonucleotides to knock down NPC1 expression in control and TNF-alpha knockout mice. In the absence of TNF-alpha, NPC1 knockdown produced liver disease with significantly less inflammation, apoptosis, and fibrosis.

In vivo antisense oligonucleotide reduction of NPC1 expression as a novel mouse model for Niemann Pick type C- associated liver disease.

UNLABELLED: Niemann-Pick type C (NPC) is a fatal autosomal recessive lipidosis that is characterized by lysosomal storage of cholesterol and glycosphingolipids. Patients exhibit prolonged neonatal jaundice, hepatosplenomegaly, and progressive neurodegeneration that generally result in death by the teen years. Most clinical cases are caused by mutations in the NPC1 gene. Current mouse models of NPC are not well suited for studying the liver disease due to the rapidly progressing neurological disease. To facilitate study of NPC-associated liver dysfunction, we have developed a novel mouse model using antisense oligonucleotides to ablate NPC1 expression primarily in the liver. Here, we show that the NPC1 knockdown leads to a liver disease phenotype similar to that of patients with NPC and the NPC(nih) mouse model. Key features include hepatomegaly, lipid storage, elevated serum liver enzymes, and increased apoptosis. CONCLUSION: This novel NPC1 antisense mouse model will allow delineation of the mechanism by which NPC1 dysfunction leads to liver cell death.