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Detection of Lung Adenocarcinoma with ROS1 Rearrangement by IHC, FISH, and RT-PCR and Analysis of Its Clinicopathologic Features

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Detection of Lung Adenocarcinoma with ROS1 Rearrangement by IHC, FISH, and RT-PCR and Analysis of Its Clinicopathologic Features Journal name: OncoTargets and Therapy Article Designation: Original Research Year: 2016 Volume: 9 OncoTargets and Therapy Dovepress Running head verso: Cao et al Running head recto: Detection of lung adenocarcinoma with ROS1 rearrangement open access to scientific and medical research DOI: http://dx.doi.org/10.2147/OTT.S94997 Open Access Full Text Article ORIGINAL RESEARCH Detection of lung adenocarcinoma with ROS1 rearrangement by IHC, FISH, and RT-PCR and analysis of its clinicopathologic features Bing Cao1–3,* Objective: To detect ROS1 rearrangement using three different assays, including Ping Wei1–3,* immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and reverse transcription Zebing Liu4 polymerase chain reaction (RT-PCR), and to analyze the clinicopathologic features of ROS1 Rui Bi1–3 rearrangement in patients with lung adenocarcinoma. Yongming Lu1–3 Methods: One hundred eighty-three consecutive patients with lung adenocarcinoma with Ling Zhang1–3 operation and follow-up data were analyzed for ROS1 rearrangement by IHC, FISH, and RT- PCR. PCR products of the RT-PCR-positive samples were sequenced for confirmation of the Jing Zhang1–3 specific fusion partners. Yusi Yang1–3 Results: Three of the 183 (1.64%) cases were identified to be positive for ROS1 rearrange- 1–3 Chen Shen ment through all three methods. The fusion patterns were CD74 e6-ROS1 e32, CD74 e6-ROS1 1–3 Xiang Du e34, and TPM3 e8-ROS1 e35, respectively. FISH-positive cases showed two types of signals, 1–3 Xiaoyan Zhou single 3′ signals (green) and split red and green signals. Using FISH as a standard method, the 1Department of Pathology, Fudan sensitivity and specificity of ROS1 IHC with +1 staining or more were 100% and 96.67%, University Shanghai Cancer Center, respectively. The sensitivity and specificity of RT-PCR were both 100%. Univariate analysis Shanghai, People’s Republic of China; 2Department of Oncology, identified female sex P( =0.044), Stage I disease (P,0.001), and ROS1-negative status (P=0.022) Shanghai Medical College, 3Institute to be significantly associated with longer overall survival. of Pathology, Fudan University, Conclusion: IHC, FISH, and RT-PCR are all effective methods for the detection of ROS1 rear- Shanghai, People’s Republic of China; 4Department of Pathology, Renji rangement. IHC would be a useful screening method in routine pathologic laboratories. RT-PCR Hospital, School of Medicine, Shanghai can detect exact fusion patterns. ROS1 rearrangement may be a worse prognostic factor. The Jiaotong University, Shanghai, People’s exact correlation of ROS1 rearrangement with prognosis and whether different fusion types are Republic of China correlated with different responses to targeted therapy need to be further investigated. *These authors contributed equally ROS1, lung adenocarcinoma, rearrangement, IHC, FISH, RT-PCR to this work Keywords: Introduction Lung adenocarcinoma is the most common histological subtype of lung cancer, which is the leading cause of cancer-related deaths worldwide.1,2 There is increasing evidence that lung adenocarcinoma could be divided into different molecular subgroups based on the identification of oncogenic drivers, such asEGFR , ALK, ROS1, RET, and MET, with unique clinicopathologic characteristics and the potential for targeted therapies.3 ROS1 is a receptor tyrosine kinase that encodes a transmembrane protein with 4 Correspondence: Xiaoyan Zhou evolutionary relationships to ALK. ROS1 fusion was originally identified in the human Department of Pathology, Fudan glioblastoma cell line U118MG in 1987.5 Recently, ROS1 fusions have been discovered University Shanghai Cancer Center, in several other tumors, including cholangiocarcinoma,6 non-small-cell lung cancer 270 Dongan Road, Shanghai 200032, People’s Republic of China (NSCLC),7–12 ovarian cancer,13 gastric carcinoma,14 and colorectal cancer.15 ROS1 Tel +86 21 6417 5590 ext 8330 fusion in NSCLC was initially identified by Rikova et al7 in 2007 using a phosphop- Fax +86 21 6417 0067 Email [email protected] roteomic screen, and ROS1 fusion was shown to participate in the formation of lung submit your manuscript | www.dovepress.com OncoTargets and Therapy 2016:9 131–138 131 Dovepress © 2016 Cao et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you http://dx.doi.org/10.2147/OTT.S94997 hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Cao et al Dovepress adenocarcinoma. Bergethon et al8 found that the features most diagnosis and histologic subtypes of lung adenocarcinoma commonly associated with ROS1-fusion NSCLC were young were made according to the 2015 World Health Organization age, never-smoking history, adenocarcinoma, and higher classification.17 The TNM stage was classified according to the tumor grade. Further studies confirmed adenocarcinoma as 2009 International Association for the Study of Lung Cancer the predominant histological type in ROS1-fusion NSCLC.9,10 staging.18 This study was approved by the Fudan University In addition, ROS1 fusion generally does not overlap with Shanghai Cancer Centre Institutional Review Board, and other known oncogenic drivers, such as EGFR mutation and conducted in accordance with the Declaration of Helsinki. ALK rearrangement.7,8,10 Written informed consent was obtained from the patients. Preclinical and clinical data have shown that ROS1 fusion cases with NSCLC are sensitive to the ALK inhibitor IHC and FISH on tissue array crizotinib.8 Crizotinib is a multitargeted kinase inhibitor, and Tissue microarrays (TMAs) containing 183 cases were built it has been approved by the US Food and Drug Administra- using 0.6 mm cores. Each tumor was sampled from two dif- tion for the treatment of patients with ALK rearrangement- ferent representative sites. TMA sections were baked and positive NSCLC. Recently, updated efficacy and safety data deparaffinized, followed by antigen retrieval with the use of for an ongoing Phase I crizotinib study (NCT00585195) indi- sodium citrate (pH =6.0). Sections were then subjected to cated that crizotinib was an effective therapy for advanced incubation with ROS1 (D4D6) rabbit monoclonal antibody ROS1-fusion NSCLC.16 And in the National Comprehensive (1:200; Cell Signaling Technology, Danvers, MA, USA) Cancer Network guidelines for NSCLC, crizotinib is listed as overnight at 4°C. Detection was conducted with EnVision+ an available targeted agent for ROS1 rearrangements. (Dako Denmark A/S, Glostrup, Denmark). The interpretation In general, ROS1 fusion occurs infrequently in lung of IHC results was conducted as described previously:19 0, no adenocarcinoma. However, given the morbidity of lung staining or nuclear expression only; 1+, faint cytoplasmic cancer, ROS1-fusion-positive patients account for a sig- staining not exceeding background in any cells; 2+, cyto- nificant number. Therefore, detection of the molecular plasmic staining exceeding background in 0%–50% of tumor alteration rapidly as well as accurately and understanding cells; and 3+, cytoplasmic staining exceeding background the tumor’s clinicopathologic features are very important in .50% of tumor cells. FISH assays were carried out utiliz- issues in the current clinical setting for the precise therapy ing a 6q22 ROS1(Tel) Spectrum Orange Probe for research of lung adenocarcinoma. In this study, we detected 183 use only (Abbott Molecular Inc, Des Plaines, IL, USA) on patients with lung adenocarcinoma at our institute to identify 4 μm thick FFPE slides. Red probes are hybridized to the 5′ ROS1 fusion-positive cases from DNA, RNA, and protein region of ROS1, and green probes to the 3′ region contain- levels by fluorescence in situ hybridization (FISH), reverse ing the tyrosine kinase domain. It was considered to be split transcription polymerase chain reaction (RT-PCR), and when red and green signals of the ROS1 break-apart probe immunohistochemistry (IHC), respectively, assessed their were physically separated by $1 signal diameter. Hybridized values in the clinical setting, and analyzed the clinicopatho- slides were stained with 4’,6-diamino-2-phenylindole and logic features. examined with a BX51 fluorescence microscope (Olympus, Tokyo, Japan). Samples were defined to be positive if. 15% Materials and methods of tumor cells presented split signals or single 3′ signals.9 Patients and tumor samples This project was conducted using data and formalin-fixed RNA extraction, RT-PCR, and sequencing paraffin-embedded (FFPE) tissue samples from Fudan Extraction of total RNA from FFPE tissue sections was University Shanghai Cancer Centre between 2007 and 2011. accomplished using the RecoverAll™ Total Nucleic Acid Patients who underwent operations and had pathologically Isolation Kit for FFPE (Thermo Fisher Scientific, Waltham, confirmed lung adenocarcinoma and follow-up data were MA, USA) following the appropriate protocols. RNA was included. Patients treated with preoperative therapy were then reverse transcribed into cDNA, using the ROS1 fusion excluded. All clinical information was gathered by
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