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H. Hager



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    MO10 - Molecular Pathology II (ID 127)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO10.07 - ALK immunohistochemistry and fluorescence in-situ hybridization in Lung adenocarcinomas from the ETOP Lungscape tumour cohort (ID 2267)

      16:50 - 16:55  |  Author(s): H. Hager

      • Abstract
      • Presentation
      • Slides

      Background
      The European Thoracic Oncology Platform LungScape database contains 2614 cases of primary resected lung carcinoma from 16 centres with patient demographics, pathological tumour data and detailed clinical follow-up. A total of 1281 cases of adenocarcinoma with >2 years clinical follow-up were selected for analysis of ALK status by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Test positive cases were matched, in order of importance at ratio 1:2, by stage, gender, smoking status, study centre, year of surgery and age with test negative cases -both for IHC and for FISH testing.

      Methods
      Testing was performed in all centres using the same protocol (IHC: Novocastra 5A4 clone antibody at 1:10 dilution, Novolink detection system. FISH: Abbott Vysis ALK break-apart probe). Each centre passed an external QA test using unknown cases in a tissue microarray before conducting the LungScape tumour testing. IHC was scored according to three intensity scores (1+, 2+, 3+) using ‘objective’ methodology previously described [1]. Maximum staining intensity was recorded. Any IHC staining was defined as IHC positive result. FISH preparations were assessed according to the Vysis protocol on all 82 IHCpositive cases plus their 164 IHCnegative matches.

      Results

      IHC cases, n=1281 FISH positive(264 tested)
      IHC negative 1199 (93.6%) 0 (0.0% of 164 controls) FISH specificity: 100%
      IHC 1+ 43 (3.35%) 2 (4.6% of IHC 1+)
      IHC 2+ 16 (1.25%) 6 (37.5% of IHC 2+)
      IHC 3+ 23 (1.8%) 20 (87% of IHC 3+)
      IHC any positive 82 (6.4%) 28 (34.1% of IHC+) FISH sensitivity: 34.1%
      FISH sensitivity was 87% for IHC 3+. IHCpositive/FISHnegative cases (n=54) were mostly IHC 1+ (75.9%), sometimes IHC 2+ (18.5%) and rarely IHC 3+ (5.5%). The frequency of never smokers was higher in the ALK IHCpositive group (29.3%) versus IHCnegative group (18.3%) {p=0.011}. Age, gender and tumour stage did not differ between IHC groups. The hazard of an event for IHCpositive cases decreases by 32% in relapse-free survival {RFS; p=0.03} and by 38% in either time-to-relapse {TTR; p=0.02} or overall survival {OS; p=0.016}. Multivariate models -adjusted for patient and tumour characteristics- indicated that IHC-ALK was a significant predictor for all three time-to-event outcomes (RFS, TTR, OS). In stratified Cox analysis, significantly higher OS was retained in the IHCpositive (HR=0.59, p=0.04) and FISHpositive (HR=0.34, p=0.03) cases in the matched cohorts, while conditional logistic regression yielded non-significant associations with 3-year survival status.

      Conclusion
      In this large cohort of surgically resected primary lung adenocarcinoma: ALK IHC positivity was 6.4%. IHC 3+ staining (prevalence 1.8%) showed 87% probability of ALK FISH positivity ALK IHC positivity was higher in never smokers and related to better clinical outcome ALK testing can be reliably implemented across multiple laboratories {1} Ruschoff et al. Virchows Arch. 2010;457(299-307).

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-023 - Anaplastic Lymphoma Kinase (ALK)-detection in Non-small Cell Lung Cancer: results of the first European IHC-based (D5F3-Optiview) panel test within 16 institutes (ID 1825)

      09:30 - 09:30  |  Author(s): H. Hager

      • Abstract

      Background
      The study was supported by Ventana Medical Systems, Inc., a Member of the Roche Group Background: The reliable identification of NSCLC patients with anaplastic lymphoma kinase (ALK) gene rearrangement is crucial for the prescription of ALK tyrosine kinase inhibitors (e.g. crizotinib). Whereas the US FDA-approval (2011) is based upon FISH-testing, the European EMA-approval (2012) refers to the definition of “ALK-positive” NSCLCs without mandating a particular test. Therefore a reliable ALK-immunohistochemistry (IHC) could be a promising option in daily routine practice.

      Methods
      Material and methods: To test the reliability of ALK-IHC-diagnosis in a multi-centre environment (17 European institutes from Belgium, Denmark, France, Germany, Scotland, Spain, Sweden and Switzerland) two tissue microarrays (TMA) consisting of 15 NSCLC cases (all adenocarcinomas; 3 cores for each case) were independently tested for ALK-expression by each laboratory using Ventana Medical System’s ALK (D5F3) primary antibody combined with OptiView DAB IHC detection and OptiView Amplification kits. Cases included in the study were unequivocal ALK-break positive or negative (by FISH), as well as so called “ALK-borderline” cases (low percentage of ALK-break positive cells by FISH, around the cut-off of 15%, therefore challenging in diagnosis, but PCR-confirmed as harbouring EML-4-ALK-fusion variants and thus eligible for therapy). Prior to the TMA-based case testing, each participating instrument was qualified using the VENTANA ALK 2 in 1 Control Slides. To provide a uniform baseline interpretation, a webinar-based training was given to all observers. This training included an overview of the ALK Interpretation Guide, a guided review of 50 patient cases using digital whole slide images, and a proficiency exam certifying each observer.

      Results
      Results: Detailed data analysis was only partly accomplished at the time of abstract submission and will be presented in detail at the “World Conference on LUNG Cancer” in Sydney. Besides the binary evaluation of the cases (ALK-negative vs. ALK-positive) observers were asked to estimate the staining intensity (0-3) within positive cases in correlation to the number of tumor cells and to generate the H-score.

      Conclusion
      Conclusion: Referring to the EMA-approval text our multi-centre study may contribute to validation and accuracy of IHC-based ALK-testing. Such a validated and reliable IHC-assay could be used: (a) as a good pre-screening method reducing time consuming and costly FISH analysis (shorten turn-around time for test results) and (b) as a final predictive approach in cases with reduced interpretability of FISH results (e.g. minimal tumor cell content in small biopsies, decalcified or artificial altered tissue, FISH in doubt/”borderline”).