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W.J. Randerath



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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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      MO10.04 - High throughput parallel amplicon sequencing of common driver mutations from FFPE lung cancer samples in molecular pathological routine diagnostics for a regional health care provider network (ID 2145)

      16:30 - 16:35  |  Author(s): W.J. Randerath

      • Abstract
      • Presentation
      • Slides

      Background
      Treatment paradigms for non–small-cell lung cancer (NSCLC) have shifted from one based only on histology to one that incorporates molecular subtypes involving particular genetic alterations such as activating mutations in EGFR or translocations of ALK. The list of therapeutically targetable lesions is rapidly increasing including mutations in genes such as EGFR, HER2, KRAS, ALK, BRAF, PIK3CA, AKT1, ROS1, NRAS, FGFR1 and MAP2K1. Analysis of these potential targets is becoming a challenge in terms of work load, tissue availability as well as cost. Within the Network Genomic Medicine Lung Cancer (NGM), a regional molecular screening network of the Center for Integrated Oncology Köln Bonn, we aimed to improve on the sequential analysis of a set of 9 target amplicons by Sanger sequencing using bench top ultra-deep parallel sequencing platforms. We aimed to reduce 1) the time requirement for comprehensive molecular diagnostics, 2) the minimal amount of formalin fixed paraffin embedded (FFPE) derived input DNA, 3) while at the same time increasing the number of target regions analysed.

      Methods
      We established a multiplex PCR to amplify up to 640 lung cancer relevant target regions from at least 20ng of FFPE derived tumor DNA. The amplicon libraries were ligated to adapters encompassing medical identifier sequences that allowed multiplexing of up to 48 patients. The resulting libraries were sequenced on a benchtop Illumina platform (MiSeq). Mutations identified by parallel sequencing were confirmed by Sanger sequencing.

      Results
      330 patients were analyzed both by traditional single PCR based Sanger sequencing of 9 amplicons and the newly established parallel sequencing protocol. We found that the NGS approach worked reliably, was less prone to sequencing analysis errors and that the time needed to complete the mutation screening was significantly reduced to 7 working days from previously 21 days. A total of at least 300ng of DNA was needed to complete the analysis of 9 amplicons by Sanger sequencing compared to 20 to 100ng of DNA needed for up to 640 amplicons analyzed by parallel sequencing.

      Conclusion
      Newly multiplex PCR based parallel sequencing allows rapid comprehensive mutation testing in routine molecular pathological diagnostics even on small FFPE embedded transbronchial biopsies.

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    MO18 - NSCLC - Targeted Therapies IV (ID 116)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO18.07 - The Network Genomic Medicine: A prospective comprehensive molecular screening network for NSCLC (ID 2898)

      16:50 - 16:55  |  Author(s): W.J. Randerath

      • Abstract
      • Presentation
      • Slides

      Background
      The potential of personalized medicine for improvement of lung cancer patient outcome has been paradigmatically shown by the treatment of advanced EGFR mutation- and ALK translocation positive NSCLC patients with the respective tyrosine kinase inhibitors. Furthermore numerous targeted drugs for molecular defined subgroups of NSCLC (e.g. ROS1- rearrangements) are in clinical development with the potential to improve outcome. Therefore one of the major challenges today is the implementation of comprehensive high-quality real time molecular diagnostics and personalized therapy for all NSCLC patients regardless of where they are treated.

      Methods
      To increase the availability of molecular testing and subsequently personalized treatment options for NSCLC patients in the catchment area of our cancer center, we established the Network Genomic Medicine (NGM) in January 2010. NGM is a collaborative network currently encompassing more than 40 different health care providers representing the full spectrum of lung cancer care in Germany including university hospitals, large non-university lung clinics and office based oncologists. NGM is based at the Center for Integrated Oncology (CIO), i.e. the joint comprehensive cancer center of the University Hospitals of Cologne and Bonn. At the NGM - headquarter genetic and clinical data are analysed and patients without approved targeted treatment options are screened for recruitment into NGM-linked personalized trials offered by the Lung Cancer Group Cologne (LCGC). Before the introduction of routine Next Generation Sequencing (NGS) within NGM in 06/2013 we screened lung adenocarcinomas (AD) via single gene assays for mutations in EGFR, KRAS, BRAF and PIK3CA, for amplifications in HER2 and translocations in ALK, ROS1 and RET. Squamous cell lung cancer (SCC) patients were screened for amplifications in FGFR1 and mutations in DDR2.

      Results
      We screened 5,145 lung cancer patients from January 2010 till April 2013. Genomic testing was feasible in 3,863 tumor samples (75%). 63% of the patients were male and 65% of samples were AD. In AD the following frequencies of genetic lesions were detected: EGFR 13.8% (288/2078); ALK 3.3% (54/1618); KRAS 33.8% (831/2457); BRAF 3.5% (76/2123); PIK3CA 3.1% (70/2190); HER2 amplified 3.6% (62/1717); RET 4.7% (4/85) and ROS1 5.1% (7/135). In SCC we found a frequency of 21% (279/1333) for FGFR1 amplification and 2.1% (11/505) for DDR2 mutations. Further we saw 18 KRAS/PIK3CA, 5 EGFR/PIK3CA, 5 BRAF/PIK3CA double mutant samples and 3 samples where a FGFR amplification was co-occurring with a DDR2 mutation. Overall 40% of NSCLC patients harboured a potentially targetable molecular alteration. In addition we could allocate more than 40 patients to early personalized clinical trials via the close collaboration of the partners within NGM and LCGC. *The frequencies of RET and ROS1 are biased, because of a preselection of pan negative patients.

      Conclusion
      NGM is one of the largest prospective molecular screening efforts for NSCLC worldwide, with currently more than 3000 samples analysed per year. Our experiences so far underline that central comprehensive high-quality real time molecular diagnostics is feasible in a large health care provider network and allows implementation of personalized medicine in routine clinical care of lung cancer patients.

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    P1.11 - Poster Session 1 - NSCLC Novel Therapies (ID 208)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P1.11-041 - Overall survival of ALK translocation - and of EGFR mutation positive NSCLC patients treated with and without personalized therapy. A retrospective analysis within the Network Genomic Medicine (ID 2916)

      09:30 - 09:30  |  Author(s): W.J. Randerath

      • Abstract

      Background
      Erlotinib, Gefitinib and Crizotinib have been approved by the European Medicines Agency (EMA) for the treatment of molecular defined patient subgroups with advanced EGFR mutation positive (EGFR M+) and ALK translocation positive (ALK +) NSCLC, respectively. In randomized clinical trials for ALK + and EGFR M+ patients comparing standard chemotherapy to TKI treatment so far no significant improvement in overall survival (OS) could be shown, based on the high crossover rate of patients initially treated in the standard chemotherapy arm into the TKI arms upon progression. Since prevention of crossover is obsolete due to ethical reasons, registry data may gain in importance for investigating the impact of new effective targeted drugs on OS in the near future.

      Methods
      Since January 2010 EGFR sequencing and ALK FISH analysis for lung adenocarcinoma was performed within the Network Genomic Medicine (NGM) as part of a broad genetic screening effort. This included mutation screening for EGFR, KRAS, BRAF and PIK3CA as well as HER2 amplification and recently also translocations of RET and ROS. Clinical and follow-up data were extracted from medical records, directly collected from physicians and patients and additionally matched with data of the Epidemiological Cancer registry of North Rhine-Westphalia, Germany.

      Results
      So far, we included a total of 44 ALK+ and 143 EGFR M+ patients into our analysis. The median age of the ALK + and EGFR M+patients was 53.5 yrs and 71 yrs, respectively. 39% of the ALK+ patients received crizotinib and 54% of the EGFR M+ patients received an EGFR TKI during the course of their disease. The median OS (mOS) of patients with an initial stage IIIb/IV was 14 months (95% CI 6.2 - 21.8) for ALK+ and 29 months (95% CI 16 - 41) for EGFR M+ patients. Both groups showed a significant difference in mOS when separated by targeted treatment status. ALK+ patients who received crizotinib had a mOS of 23 months (95% CI 12.2 - 33.8) and patients who did not receive crizotinib had a mOS of 8 months (95% CI 0.0 - 17.4) (p = 0.01). EGFR M+ patients who received an EGFR TKI had a mOS of 31 months (95% CI not computable) and patients who did not receive an EGFR TKI had a mOS of 9 months (95% CI 4.9 - 13.1) (p < 0.001). There were no significant differences with regard to treatment of a platinum-containing chemotherapy, age or sex between the two groups.

      Conclusion
      Screening patients for genetic driver mutations identified patients with EGFR mutations and ALK translocations that were not treated with a kinase inhibitor. Comparing these cohorts of patients that only received standard chemotherapy to those subsequently treated with a personalized approach showed a significant improvement in OS. This confirms the predictive value of ALK translocations and EGFR mutations for treatment with the respective TKIs