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L. Morrison

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    MINI 22 - New Technology (ID 134)

    • Event: WCLC 2015
    • Type: Mini Oral
    • Track: Biology, Pathology, and Molecular Testing
    • Presentations: 1
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      MINI22.03 - Next Generation Immunohistochemical Stains; True Multiplex (Quadruple) Immunohistochemical Panel for Non-Small Cell Lung Carcinoma (ID 2119)

      16:45 - 18:15  |  Author(s): L. Morrison

      • Abstract
      • Presentation
      • Slides

      Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung cancers, the vast majority of which are non-small cell lung carcinomas (NSCC). It is critical to further distinguish adenocarcinomas from squamous carcinomas in order to optimize the efficiency of the precision medicine analysis for the detection of active molecular targets for therapies. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin-A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin-A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung.

      Developmental reagents from Ventana Medical Systems, Inc. were leveraged for this study. Detection of CK 5/6 and p40 [BC28] was used to identify squamous cell carcinoma cells. Detection of Napsin A and TTF-1 was used to identify adenocarcinoma cells. Detection was accomplished using secondary antibody:enzyme conjugates and orthogonal chromogenic detection chemistries to simultaneously detect all 4 biomarkers. Fully automated multiplexed detection was performed on a Benchmark XT with 4 microns thick sections from formalin fixed paraffin embedded, non-small cell lung cancer specimens obtained from both the Ventana Medical Systems, Inc. tissue bank and from the Thomas Jefferson University’s Department of Pathology, Anatomy and Cell Biology laboratories. Detection of each marker in multiplex was compared to individual detections using diaminobenzidine deposition according to established Ventana Medical Systems, Inc. protocols. All detections were reviewed by a board certified pathologist.

      Adenocarcinomas (7 of 7) and the adenocarcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for TTF-1 (yellow nuclear stain) and Napsin-A (pink cytoplasmic granular stain). Squamous cell carcinomas (5 of 5) and the squamous cell carcinoma components of the adenosquamous carcinomas (6 of 6) stained positive for p40 (blue nuclear stain) and CK5/6 (brown cytoplasmic stain). The colors were clear, distinct, easily differentiated and recognizable. There was no discrepancy between the expression of the individual antibodies and the expression of the same antibodies in the multiplex setting.

      Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or IHC, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. This new quadriplex IHC offers the capability with a single 4 micron section to accurately diagnose primary lung adenocarcinoma, squamous cell carcinoma or adenosquamous carcinoma and while conserving tissue for additional molecular testing.

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