Virtual Library

 x 
My Library Virtual Library FAQ

Start Your Search

R.R. Tubbs



Author of

  • +

    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
    • +

      O04.06 - An international standardization study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators (ID 2875)

      10:30 - 12:00  |  Author(s): R.R. Tubbs

      • Abstract
      • Presentation
      • Slides

      Background
      The goal of personalized medicine is treating patients with a therapy predicted to be efficacious based on the molecular characteristics of the tumor, thereby sparing the patient futile or detrimental therapy. Anaplastic lymphoma kinase (ALK) inhibitors are effective against ALK positive non-small cell lung cancer (NSCLC) tumors, but to date the only US Food and Drug Administration approved companion diagnostic is a break-apart fluorescence in situ hybridization (FISH) assay. Immunohistochemistry (IHC) is a clinically applicable cost-effective test that is sensitive and specific for ALK protein expression. The purpose of this study was to assemble an international team of expert pathologists to evaluate and standardize the interpretation of a new automated standardized ALK IHC assay.

      Methods
      Archival NSCLC tumor specimens (n=103) previously tested for ALK rearrangement by FISH were provided by the international collaborators. These specimens were stained by IHC with the anti-ALK (D5F3) primary antibody (Ventana Medical Systems, Inc) combined with OptiView DAB IHC detection and OptiView amplification (Ventana Medical Systems, Inc). The evaluators went through an interpretation training session and scored the specimens as positive, if strong granular cytoplasmic brown staining was present in tumor cells, or negative. IHC results were compared to the FISH results and inter-evaluator agreement comparisons made.

      Results
      Overall for the 100 evaluable cases the ALK IHC assay was highly sensitive (90%), specific (95%) and accurate (93%) relative to the ALK FISH results. Similar results were observed using a majority score. For the discrepant cases IHC negativity was scored by 7/7 on 3 FISH positive cases and 6/7 evaluators on 2 additional FISH positive cases. IHC positivity was scored on 2 FISH negative cases by 7/7 readers. There was agreement among 7/7 and 6/7 readers on 88% and 96%% of the cases before a consensus review, respectively, and following a review there was agreement among 7/7 and 6/7 on 95% and 97% of the cases, respectively.

      Conclusion
      Based on expert evaluation the ALK IHC assay using the D5F3 antibody combined with Optiview Detction and Optiview amplification is sensitive, specific and accurate, relative to FISH, and a majority score of multiple readers does not improve these results over an individual reader’s score. Excellent inter-reader agreement was observed for the IHC assay. These data support the algorithmic use of ALK IHC as a screening procedure for ALK protein expression in NSCLC.

      Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.

      Only Active Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login or select "Add to Cart" and proceed to checkout.

  • +

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
    • +

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

      09:30 - 16:30  |  Author(s): R.R. Tubbs

      • 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.