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  P3.06 - Poster Session 3 - Prognostic and Predictive Biomarkers (ID 178)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12467

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    P3.06-027 - A comparison of FISH and immunohistochemistry in the detection of ALK rearrangement in lung adenocarcinoma (ID 2392)

    09:30 - 09:30  |  Author(s): A. Wotherspoon
    • Abstract

    Background
    Personalised treatments for lung cancer are being increasingly employed. Around 4% of pulmonary adenocarcinomas have rearrangements of the ALK gene resulting in oncogenic fusion proteins. Such cases are sensitive to the small molecule tyrosine kinase inhibitor Crizotinib, and detection of the ALK rearrangement is crucial to the treatment of these patients. Currently the only approved test for the detection of ALK-positive NSCLC is FISH. Various immunohistochemical assays have been proposed as alternatives or screening tools, which are cheaper, quicker and easier to perform and interpret than FISH. Validation of these immunohistochemical tests is therefore important.

    Methods
    15 FISH positive cases were obtained from local archives at the Royal Marsden and Royal Brompton Hospitals, with accompanying data on crizotinib therapy and clinical response. A further 14 FISH negative and FISH uncertain cases were also retrieved. 3 antibodies were optimised for immunohistochemical detection of ALK: D5F3, marketed by Ventana and using their proprietary automated system, ALK1 (Dako), and 5A4 (Abcam). All three antibodies were applied to sections from the archival cases. Antibodies were semiquantitatively scored on their intensity (0-3) and proportion of malignant cells stained (0-100%). Cutoffs for positivity/negativity were set by ROC analysis to optimise correct classification.

    Results
    Positivity according to the Vysis FISH assay (i.e. appropriately abnormal FISH signal in at least 15% of cells) was taken as the gold standard. The Ventana assay (D5F3) was markedly more intense but showed higher background than the other two antibodies. ROC analysis of staining intensity showed that the Ventana and Dako antibodies should be considered positive when staining is of 'moderate' intensity or above. The Abcam assay was discriminatory when any positivity was seen. Scoring of proportion did not improve specificity or sensitivity with any of the antibody tests. Subsequent analyses were of intensity scoring. Taking all cases together, all 3 antibodies were highly specific (100%) and sensitive (Ventana 86%, Abcam 79% and Dako 71%). In excision specimens, all 3 assays showed sensitivity of 83%. In cytology and small biopsy specimens, Ventana performed the best (specificity of 88% compared to 75% for Dako and 63% for Abcam) although the numbers are small. No ‘false positive’ (-ve FISH, +ve IHC) cases were seen; the only disparity between FISH and IHC were 'false negative' cases. Two cases were FISH-positive but universally IHC-negative (with all 3 assays). Of these, one was treated with crizotinib, and failed to respond.

    Conclusion
    IHC is a highly specific (100%) and sensitive (71%-86%) test for ALK rearrangement in archival FFPE tumour tissue. In this study of 15 ALK-rearranged tumours, the Ventana D5F3 assay performed the best, despite having relatively high background staining. Occasional cases are FISH-positive but IHC-negative. One such case was not responsive to Crizotinib, further supporting IHC as a test predictive of drug response. IHC has the potential to replace FISH for the detection of candidates for Crizotinib therapy. However, further work is required to direct the treatment of tumours with discordant FISH and immunohistochemical tests.