Screening of anaplastic lymphoma kinase rearrangement by immunohistochemistry in non-small cell lung cancer: correlation with fluorescence in situ hybridization

J Thorac Oncol. 2011 Mar;6(3):466-72. doi: 10.1097/JTO.0b013e31820b82e8.


Background: The use of a standard immunohistochemistry (IHC) assay to detect the anaplastic lymphoma kinase (ALK) protein in lung cancer is challenging. There are no universally accepted, evidence-based guidelines on identifying patients with ALK-rearranged lung cancer using IHC.

Methods: We retrospectively reviewed 465 resected specimens of non-small cell lung cancer using a tissue microarray as a test set. ALK protein expression using IHC with 5A4 monoclonal antibody (Novocastra) and ALK gene rearrangement using fluorescence in situ hybridization (FISH) with dual-color break-apart probes (Abbott molecular) were examined. Immunoreactivity was scored as 0, 1, 2, or 3, and the results were compared with the FISH results. A diagnostic algorithm was derived from the correlation of the IHC and FISH results and applied to an additional 187 adenocarcinoma samples used as a validation set.

Results: In the test set, ALK protein expression was detected in 40 patients (40/465, 8.6%), consisting of IHC scores of 1 (n = 14), 2 (n = 10), and 3 (n = 16), whereas 19 patients (19/453, 4.2%) were FISH-positive. All the FISH-positive patients were assigned IHC scores of 2 or 3. All the patients with ALK IHC scores of 3 were FISH-positive, those with scores of 0 or 1 were FISH-negative, and those with scores of 2 were FISH variable. In the validation set, ALK protein expression was detected in 14 patients (scores of 1, n = 2; scores of 2, n = 6; and scores of 3, n = 6), of which nine patients (9/187, 4.8%) were FISH-positive. All the patients with IHC scores of 0 or 1 were FISH-negative, and those with scores of 3 were FISH- positive. Among the patients with IHC scores of 2, three (3/6, 50%) were FISH-positive.

Conclusions: The sensitivity and specificity of IHC was 100% and 95.8%, respectively. These data supported an IHC scoring algorithm in which ALK IHC scores of 0, 1, or 3 were highly compatible with FISH results, and IHC scores of 2 were variable. Based on these findings, the IHC assay using the 5A4 antibody reliably detected non-small cell lung cancer with ALK rearrangement and may be useful as a screening method to identify these tumors.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenocarcinoma / diagnosis
  • Adenocarcinoma / genetics
  • Adenocarcinoma / metabolism
  • Adult
  • Aged
  • Aged, 80 and over
  • Anaplastic Lymphoma Kinase
  • Carcinoma, Large Cell / diagnosis
  • Carcinoma, Large Cell / genetics
  • Carcinoma, Large Cell / metabolism
  • Carcinoma, Non-Small-Cell Lung / diagnosis*
  • Carcinoma, Non-Small-Cell Lung / genetics
  • Carcinoma, Non-Small-Cell Lung / metabolism
  • Carcinoma, Squamous Cell / diagnosis
  • Carcinoma, Squamous Cell / genetics
  • Carcinoma, Squamous Cell / metabolism
  • Cohort Studies
  • Female
  • Follow-Up Studies
  • Gene Rearrangement*
  • Humans
  • Immunoenzyme Techniques
  • In Situ Hybridization, Fluorescence*
  • Lung Neoplasms / diagnosis*
  • Lung Neoplasms / genetics
  • Lung Neoplasms / metabolism
  • Male
  • Middle Aged
  • Neoplasm Staging
  • Prognosis
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Retrospective Studies
  • Sensitivity and Specificity
  • Tissue Array Analysis
  • Young Adult


  • ALK protein, human
  • Anaplastic Lymphoma Kinase
  • Receptor Protein-Tyrosine Kinases