title: Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended.
abstract: OBJECTIVE
  Immunohistochemistry (IHC) has become a promising method for pre-screening ALK-rearrangements in non-small cell lung carcinomas (NSCLC). Various ALK antibodies, detection systems and automated immunostainers are available. We therefore aimed to compare the performance of the monoclonal 5A4 (Novocastra, Leica) and D5F3 (Cell Signaling, Ventana) antibodies using two different immunostainers. Additionally we analyzed the accuracy of prospective ALK IHC-testing in routine diagnostics.
  
  
  METHODS
  Seventy-two NSCLC with available ALK FISH results and enriched for FISH-positive carcinomas were retrospectively analyzed. IHC was performed on BenchMarkXT (Ventana) using 5A4 and D5F3, respectively, and additionally with 5A4 on Bond-MAX (Leica). Data from our routine diagnostics on prospective ALK-testing with parallel IHC, using 5A4, and FISH were available from 303 NSCLC.
  
  
  RESULTS
  All three IHC protocols showed congruent results. Only 1/25 FISH-positive NSCLC (4%) was false negative by IHC. For all three IHC protocols the sensitivity, specificity, positive (PPV) and negative predictive values (NPV) compared to FISH were 96%, 100%, 100% and 97.8%, respectively. In the prospective cohort 3/32 FISH-positive (9.4%) and 2/271 FISH-negative (0.7%) NSCLC were false negative and false positive by IHC, respectively. In routine diagnostics the sensitivity, specificity, PPV and NPV of IHC compared to FISH were 90.6%, 99.3%, 93.5% and 98.9%, respectively.
  
  
  CONCLUSIONS
  5A4 and D5F3 are equally well suited for detecting ALK-rearranged NSCLC. BenchMark and BOND-MAX immunostainers can be used for IHC with 5A4. True discrepancies between IHC and FISH results do exist and need to be addressed when implementing IHC in an ALK-testing algorithm.
authors.affiliation: Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland. Electronic address: spasenija.savicprince@usb.ch.
authors.forename: Spasenija
authors.initials: S
authors.lastname: Savic
authors.affiliation: Institute of Pathology, Cantonal Hospital Lucerne, 6000 Luzern 16, Lucerne, Switzerland. Electronic address: Joachim.Diebold@luks.ch.
authors.forename: Joachim
authors.initials: J
authors.lastname: Diebold
authors.affiliation: Institute of Surgical Pathology, University Hospital Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland. Electronic address: ak.zimmermann@gmx.de.
authors.forename: Anne-Katrin
authors.initials: AK
authors.lastname: Zimmermann
authors.affiliation: Institute of Pathology, Cantonal Hospital St. Gallen, Rorschacher Strasse 95, 9007 St. Gallen, Switzerland. Electronic address: Wolfram.Jochum@kssg.ch.
authors.forename: Wolfram
authors.initials: W
authors.lastname: Jochum
authors.affiliation: Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland. Electronic address: betty.baschiera@usb.ch.
authors.forename: Betty
authors.initials: B
authors.lastname: Baschiera
authors.affiliation: Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland. Electronic address: susanne.grieshaber@usb.ch.
authors.forename: Susanne
authors.initials: S
authors.lastname: Grieshaber
authors.affiliation: Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland. Electronic address: luigi.tornillo@unibas.ch.
authors.forename: Luigi
authors.initials: L
authors.lastname: Tornillo
authors.affiliation: Institute of Pathology, University Hospital Lausanne, Rue du Bugnon 25, 1011 Lausanne, Switzerland. Electronic address: Bettina.Bisig@chuv.ch.
authors.forename: Bettina
authors.initials: B
authors.lastname: Bisig
authors.affiliation: Department of Pathology, Aberdeen Royal Infirmary, Foresterhill, Aberdeen AB25 2ZD, UK. Electronic address: k.kerr@abdn.ac.uk.
authors.forename: Keith
authors.initials: K
authors.lastname: Kerr
authors.affiliation: Institute of Pathology, University Hospital Basel, Schoenbeinstrasse 40, 4031 Basel, Switzerland. Electronic address: lukas.bubendorf@usb.ch.
authors.forename: Lukas
authors.initials: L
authors.lastname: Bubendorf
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D014408:Biomarkers, Tumor; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism*; D055088:Early Detection of Cancer; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics* / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; D016283:Proto-Oncogene Proteins p21(ras)
keywords: 5A4; ALK; D5F3; FISH; Immunohistochemistry; Lung cancer