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. 2012 Sep 1;18(17):4570-9.
doi: 10.1158/1078-0432.CCR-12-0550. Epub 2012 Aug 23.

Identifying and Targeting ROS1 Gene Fusions in Non-Small Cell Lung Cancer

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Free PMC article

Identifying and Targeting ROS1 Gene Fusions in Non-Small Cell Lung Cancer

Kurtis D Davies et al. Clin Cancer Res. .
Free PMC article

Abstract

Purpose: Oncogenic gene fusions involving the 3' region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non-small cell lung cancer (NSCLC) and establish the fusion proteins as drug targets.

Experimental design: An NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by FISH. This assay was also used to screen patients with NSCLC. In positive samples, the identity of the fusion partner was determined through inverse PCR and reverse transcriptase PCR. In addition, the clinical efficacy of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2-ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and Western blotting.

Results: In the TMA panel, 5 of 428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. In addition, 1 of 48 patients tested positive for rearrangement, and this patient showed tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4-ROS1 fusion, whereas two TMA samples expressed the CD74-ROS1 fusion and two others expressed the SLC34A2-ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was antiproliferative and downregulated signaling pathways that are critical for growth and survival.

Conclusions: ROS1 inhibition may be an effective treatment strategy for the subset of patients with NSCLC whose tumors express ROS1 fusion genes.

Figures

Figure 1
Figure 1. ROS1 FISH and RT-PCR analysis of NSCLC TMA panel
(A) ROS1 rearrangement-positive samples analyzed by break-apart FISH assay. Red probes are hybridized to the 5’ region of ROS1 and green probes to the 3’ region. Yellow arrows denote fused signals (non-rearranged ROS1 allele), while green and red arrows denote 3’ and 5’ regions of ROS1, respectively, that have been separated by rearrangement. Left image demonstrates split signal pattern (patient #1), while right image demonstrates single-green signal pattern (patient #4). (B) Agarose gel electrophoresis of RT-PCR products from ROS1 rearrangement-positive TMA samples (along with samples from HCC78 cells and CU patient for comparison). The approximate expected positions of long transcripts (fusion to ROS1 exon 32), short transcripts (fusion to ROS1 exon 34), and primer dimers (PD), are shown. In all cases, amplified DNA was gel isolated and sequenced (data not shown).
Figure 2
Figure 2. SDC4-ROS1 fusion identified in an NSCLC patient who responded to crizotinib treatment
(A) Break-apart FISH analysis of a NSCLC patient’s tumor sample demonstrating rearrangement of the ROS1 gene. Red probes are hybridized to the 3’ region of ROS1 and green probes to the 5’ region (note that colors are reversed compared to Fig. 1). Yellow arrows denote fused signals (non-rearranged ROS1 allele), while red and green arrows denote 3’ and 5’ regions of ROS1, respectively, that have been separated by rearrangement. (B) Sequencing of products from RT-PCR performed on patient tumor sample using primers specific for SDC4 and ROS1. Upper chromatogram identifies the break point in the long transcript and lower chromatogram identifies the break point in the short transcript. (C) Left - IL3-independent growth of Ba/F3 cells transduced with empty vector or a SDC4-ROS1(exon 32) construct (SD2:R32). Data is reported as the percent of day 0. Values represent the mean ± SEM (n=3). Right – western blots of cell lysates from empty vector and SD2:R32 transduced Ba/F3 cells. (D) Representative FDG18 PET-CT images before and after crizotinib treatment showing marked decrease in the size of the right lung mass and pleural disease with concomitant decrease in the standard uptake value (SUV).
Figure 3
Figure 3. ROS1 inhibition is anti-proliferative in cells expressing ROS1 gene fusions
(A) Ba/F3 cells transduced with an SDC4-ROS1(exon 32) construct (SD2:R32) or an EML4-ALK(exon 20) construct (E6:A20) were treated with dose ranges of NVP-TAE684 (left) or crizotinib (right) for 3 days in the absence of IL3 and then analyzed by MTS assay. Values represent the mean ± SEM (n=4). Calculated IC50 values were as follows: NVP-TAE684 - SD2:R32 (12.3nM), E6:A20 (2.4nM), crizotinib - SD2:R32 (31nM), E6:A20 (83nM). (B) HCC78 cells were treated with dose ranges of NVP-TAE684 or crizotinib for 3 days and then analyzed by MTS assay. Values represent the mean ± SEM (n=5). Calculated IC50 values were 105nM for NVP-TAE684 and 775nM for crizotinib. (C) HCC78 cells were treated with dose ranges of NVP-TAE684 (left) or crizotinib (right) for 24 hours and then stained with propidium iodide. Cell cycle distribution was then assessed by flow cytometry. Results are representative of 2 independent experiments. (D) HCC78 cell lysates were analyzed by phospho-RTK array. The positions of phospho-EGFR and phospho-MET spots are indicated. Un-labeled spots at the four corners of the array are the positive control. (E) Left - HCC78 cells were treated with dose ranges of gefitinib (EGFR kinase inhibitor) or PF-04217903 (MET kinase inhibitor) for 3 days and then analyzed by MTS assay. Values represent averages of 2 independent experiments. Right - HCC78 cells were treated with a dose range of NVP-TAE684 alone or in the presence of 1uM gefitinib or 1uM PF-04217903 for 3 days and then analyzed by MTS assay. Values represent the mean ± SEM (n=4). Calculated IC50 values were 157nM for +vehicle, 43nM for +1uM gefitinib, and 141nM for +1uM PF-04217903. +1uM gefitinib was less than +vehicle and +1uM PF-04217903 as determined by one-way ANOVA analysis followed by Bonferroni’s multiple comparison test (p<0.05).
Figure 4
Figure 4. ROS1 inhibition down-regulates proliferation and survival pathways in HCC78 cells
HCC78 cells were treated with NVP-TAE684, crizotinib, or PF-04217903 at the indicated concentrations for 4–6 hours. Lysates of the cells were then analyzed by western blot using the indicated antibodies. Results are representative of two independent experiments.

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