title: Comparing four different ALK antibodies with manual immunohistochemistry (IHC) to screen for ALK-rearranged non-small cell lung cancer (NSCLC).
abstract: OBJECTIVE
  Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) screening is essential to its treatment such as crizotinib. Different assays have been developed to detect ALK rearrangements, such as fluorescence in situ hybridization (FISH), reverse transcriptase-PCR (RT-PCR), and immunohistochemistry (IHC). However, ALK detection has not been applied widely in all hospitals. Moreover, IHC has been proposed to be a pre-screening tool because of its wide application in clinics. Since the low expression of ALK protein, the sensitivity and specificity of ALK antibody are the keys to the success of IHC screening. Therefore, we compared different antibodies to find the best one for IHC detection.
  
  
  METHODS
  We evaluated ALK expression by four different ALK antibodies: clone D5F3 (Ventana), clone D5F3 (CST), clone 1A4/1H7 (OriGene Tech.), and clone 5A4 (Abcam) based on manual IHC in a cohort of 60 NSCLCs. The results were compared with those from automated IHC (clone D5F3, Ventana). All cases were evaluated independently by ALK FISH.
  
  
  RESULTS
  32 ALK-positive and 28 ALK-negative NSCLCs were identified by automated IHC (D5F3, Ventana) and FISH analysis. Based on conventional manual IHC, the sensitivity of four antibodies-D5F3 (Ventana), D5F3 (CST), 1A4/1H7 (OriGene Tech.), and 5A4 (Abcam)-was 93.8%, 84.4%, 93.8%, and 56.3%, respectively. Their specificities and positive predictive values were 100%. The percentage of strong-moderate staining was 65.6%, 62.5%, 68.8%, and 21.9%, respectively. Compared with automated IHC (D5F3, Ventana), each staining concordance was 96.7%, 91.7%, 96.7%, and 76.7%, respectively, and each presented staining heterogeneity (weak-moderate-strong intensity).
  
  
  CONCLUSIONS
  These data indicated that manual IHC with a more reliable ALK antibody might provide an effective strategy for screening ALK gene rearrangements in all NSCLC patients, followed by confirmatory FISH analysis in IHC-positive cases.
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China. Electronic address: qinshen2005@163.com.
authors.forename: Qin
authors.initials: Q
authors.lastname: Shen
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Xuan
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Shanshan
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Biao
authors.initials: B
authors.lastname: Liu
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Yanfen
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Qiuyuan
authors.initials: Q
authors.lastname: Xia
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China.
authors.forename: Qiu
authors.initials: Q
authors.lastname: Rao
authors.affiliation: Department of Pathology, Jinling Hospital, Clinical Medical School of Southern Medical University, 305 East Zhongshan Road, Nanjing 210002, PR China. Electronic address: zhouxj3456@126.com.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Zhou
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D015321:Gene Rearrangement; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism*; D008297:Male; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*
chemical_list: C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases
keywords: Anaplastic lymphoma kinase (ALK) gene rearrangement; Antibody 1A4/1H7; Immunohistochemistry (IHC); Non-small cell lung cancer (NSCLC)