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A. Soltermann



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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): A. Soltermann

      • 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: 2
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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): A. Soltermann

      • 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”).

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      P1.06-055 - The RON (MST1R)/MSP pathway is a potential therapeutic target in malignant plural mesothelioma (ID 3250)

      09:30 - 09:30  |  Author(s): A. Soltermann

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer. Treatment options are limited and drug resistance is common. Thus, there is a need to identify novel therapeutic targets in this disease in order to improve treatment options and survival times. Macrophage stimulating protein (MSP) is the only ligand recognised to bind to the RON receptor (MST1R). RON is a member of the MET proto-oncogene family. The MSP-RON signalling pathway has been implicated in a variety of cellular functions such as macrophage morphogenesis and phagocytosis. De-regulation of this pathway has been linked to tumour progression and metastasis in a number of cancers. We have previously identified RON as frequently activated in MPM and high positivity for RON by IHC was an independent predictor of favourable prognosis.

      Methods
      A panel of mesothelioma cell lines were screened for the expression of MSP and RON at the mRNA (RT-PCR) and protein (Western blot) level. The effect of MSP, IMC-RON8 (a humanised IgG1 monoclonal antibody), LCRF004 (a small molecule inhibitor) and NRWHE (a small peptide) was examined in the H226 cell line using proliferation (BrdU ELISA), apoptosis (Multi-parameter apoptosis assay) and migration assays (xCELLigence). A phospho-kinase proteome profiler array was utilised to detect the downstream signalling pathways activated upon MSP stimulation. The expression of MSP and the macrophage marker, CD-68, was examined by IHC using MPM TMAs. Studies are ongoing to determine the effect of the LCRF004 compound in vivo using a xenograft murine model with the H226 cells.

      Results
      The mRNA and protein levels of RON and MSP were differentially expressed in a panel of MPM cell lines. Treatment with LCRF004 resulted in significantly decreased proliferation and increased apoptosis in the H226 cells. MSP was unable to rescue the cells from the effects of LCRF004. NRWHE and RON8 had little effect on either proliferation or apoptosis. All of the compounds examined inhibited the migration capacity of the H226 cells. The combination of LCRF004 and MSP produced a synergistic effect, showing greater inhibition of migration than either compound alone. However, MSP treatment resulted in the up-regulation of a number of phosphor-kinases including Akt, ERK and the Src family. Currently, a number of proteins identified in the array studies are undergoing validation. Results of an in vivo H226 murine model using the LCRF004 compound will be presented at the meeting.

      Conclusion
      From previous work performed in this laboratory, we have determined that high expression of RON in MPM is an independent predictor of favourable prognosis. IHC was performed on a TMA of MPM patient samples and high expression levels of MSP correlated with better survival. There was no association between CD68 staining and MSP, nor correlation of CD68 expression with survival. Targeting the RTK domain of the RON receptor with a small molecule inhibitor is an effective interventional strategy in MPM. The seemingly counter intuitive results obtained from the MPM TMA studies and the in vitro experimental data, may be RON isoform dependant. Additional studies are ongoing to further delineate the RON-MSP axis in MPM.

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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
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      P3.06-020 - Prognostic Markers for Mesothelioma Patients Treated with Induction Chemotherapy Followed by Extrapleural pneumonectomy (ID 1879)

      09:30 - 09:30  |  Author(s): A. Soltermann

      • Abstract

      Background
      Multimodal treatment provides the best outcome for some but not all malignant pleural mesothelioma (MPM) patients. Therefore the identification of prognostic markers helping to select patients remains a subject of key importance. Frequent loss of NF2 tumor suppressor gene, a regulator of Hippo pathway and mammalian target of rapamycin (mTOR) has been well documented in MPM. We recently observed loss of expression of phosphatase and tensin homologue (PTEN), a tumor suppressor gene of the phosphatidylinositol 3-kinase (PI3K)/mTOR pathway, in 62% of MPM patients. In this regard, increased activity of these pathways stemming from loss of abovementioned tumor suppressor genes may serve as potential prognosticator as well as therapeutic target. Here we aim to assess prognostic significance of PI3K/mTOR and Hippo pathways for MPM patients treated with a multimodal approach.

      Methods
      Large cohort of MPM patients uniformly treated with induction chemotherapy followed by extrapleural pneumonectomy was employed in this study. Tissue micro arrays were constructed composing of paired samples from patients (n = 153) collected pre- and post-induction chemotherapy. All tissues were evaluated for apoptotic index (cleaved caspase-3) and proliferation index (Ki-67). Expression levels of biomarkers of PI3K/mTOR (PTEN, p(phosphorylated)-mTOR and p-S6) and Hippo signaling pathway (nuclear-YAP and nuclear-Survivin) were evaluated by immunohistochemistry and correlated with overall survival (OAS) and progression free survival (PFS).

      Results
      Survival analysis showed that high p-S6 expression and high Ki-67 index in samples of treatment naïve patients was associated with shorter PFS (p = 0.02 and p = 0.04, respectively). High Ki-67 index after chemotherapy remained associated with shorter PFS (p = 0.03) and OAS (p = 0.02). Paired comparison of marker expression in samples prior and post induction chemotherapy revealed that decreased cytoplasmic PTEN as well as increased p-mTOR expression was associated with a worse OAS (p = 0.04 and 0.03, respectively). No correlation was observed between expression level of nuclear-YAP with PFS or OAS.

      Conclusion
      Ki-67 proliferation index has prognostic value for MPM patients treated with multimodality approach in when analyzed both pre- and post- induction chemotherapy specimens. Our results further reveal the prognostic significance of expression changes of PI3K/mTOR pathway components during induction chemotherapy. If confirmed, these data suggest PI3K/mTOR pathway to be a target for selected MPM patients.