Author of

• 11745

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  P2.11 - Poster Session 2 - NSCLC Novel Therapies (ID 209)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Medical Oncology
  • Presentations: 1
  • Moderators:
  • Coordinates: 10/29/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 11746

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    P2.11-001 - <em>ALK</em> Genotyping via Liquid Based ThinPrep Slides Prepared from Fine Needle Aspirates in NSCLC (ID 258)

    09:30 - 09:30  |  Author(s): F. Almeida
    • Abstract

    Background
    Rearrangements at chromosome locus 2p23 encompassing the Anaplastic Lymphoma Kinase (ALK) gene in non-small cell lung carcinomas (NSCLC) drive expression of the oncogenic ALK encoded protein and provide a target for crizotinib and other ALK inhibitors. A companion diagnostic assay, fluorescence in-situ hybridization (FISH) using conventional formalin-fixed paraffin-embedded (FFPE) tissue samples, was approved for identifying patients eligible for treatment and has been the commonly employed method for identification of ALK rearrangements. The FISH assay assessing ALK rearrangments may fail due to absence of enumerable FISH signals, insufficient number of tumor cells, or autofluorescence obscuring specific FISH signals, especially for cytoblocks obtained as part of staging or diagnostic fine needle aspiration (FNA) biopsy. FISH performed on liquid based ThinPrep slides (ThinPrepFISH) may represent a robust alternative to conventional FFPE-FISH.

    Methods
    ThinPrep slides obtained by FNA biopsy from 156 patients with advanced NSCLC were evaluated at Cleveland Clinic by ThinPrep-FISH using a standard ALK break-apart two color probe set (Abbott Molecular Vysis, Des Plaines; AMV). Cell pellets were not available for preparation of FFPE cytoblocks from the cell pellet in 7/156 (4%), and the FFPE cytoblock slides did not contain tumors cells for evaluation in 33/156 (21%) despite the presence of abundant tumor cells in the ThinPrep slide. Given the failure rate for FFPE-FISH using cytoblocks (>30% in our preliminary experience) we chose to use ultrasensitive immunohistochemistry (IHC) for ALK expression in corresponding cytoblocks {D5F3 antibody (Cell Signaling) coupled with enhanced detection sensitivity (OptiView with signal amplification (Ventana; Tucson)} as the reference data set [J Mol Diagn 2013 May; 15(3):341-6] The same ThinPrep slide used for cytopathology diagnosis was probed using ThinPrep-FISH. Specific areas of the ThinPrep-FISH slide having abundant tumor cells were etched with a diamond tip pen on the reverse side of the slide prior to removal of the cover slip; all areas of the ThinPrep slide were screened qualitatively and signals enumerated selectively for tumor cells. Interpretative criteria used for ThinPrepFISH were the same criteria as is used for the IVD AMV probe set.

    Results
    ThinPrepFISH ALK signals were robust and not compromised by nuclear truncation inherent in FFPE tissue FISH in 155 / 156 cases. One of 156 cases displayed no signals, but was probed 11 months after the ThinPrep slide had been prepared. Overall, 9/156 cases (6%) were ALK rearranged. For 116 ThinPrep slides successfully paired with FFPE cytoblocks containing tumor cells, 7/116 cases (6%) were ThinPrep-FISH positive and IHC positive for ALK rearrangements/expression, 2/116 cases (1.7%) were ThinPrep-FISH positve but IHC negative, and 107/116 cases were ThinPrep-FISH negative and IHC negative (sensitivity 100%, specificity 98.2%, overall agreement 98.3%).

    Conclusion
    Detection of ALK gene rearrangements in liquid cytology ThinPrep slides prepared from fine needle aspiration cytopathology specimens derived from patients with NSCLC can be confidently used for clinical ALK molecular testing.