Screening for ALK in non-small cell lung carcinomas: 5A4 and D5F3 antibodies perform equally well, but combined use with FISH is recommended

Lung Cancer. 2015 Aug;89(2):104-9. doi: 10.1016/j.lungcan.2015.05.012. Epub 2015 May 21.


Objectives: 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.

Materials and 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.

Keywords: 5A4; ALK; D5F3; FISH; Immunohistochemistry; Lung cancer.

MeSH terms

  • Anaplastic Lymphoma Kinase
  • Biomarkers, Tumor
  • Carcinoma, Non-Small-Cell Lung / diagnosis
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Non-Small-Cell Lung / metabolism*
  • Early Detection of Cancer
  • Humans
  • Immunohistochemistry
  • In Situ Hybridization, Fluorescence
  • Lung Neoplasms / diagnosis
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / metabolism*
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Receptor Protein-Tyrosine Kinases / genetics*
  • Receptor Protein-Tyrosine Kinases / metabolism*
  • Reproducibility of Results
  • Retrospective Studies
  • Sensitivity and Specificity


  • Biomarkers, Tumor
  • KRAS protein, human
  • ALK protein, human
  • Anaplastic Lymphoma Kinase
  • Receptor Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins p21(ras)