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M. Gardizi



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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): M. Gardizi

      • 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): M. Gardizi

      • 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: 3
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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): M. Gardizi

      • 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

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      P1.11-042 - SORAVE: Sorafenib and everolimus for patients with solid tumors and with KRAS mutated NSCLC - results of a phase I study. (ID 3068)

      09:30 - 09:30  |  Author(s): M. Gardizi

      • Abstract

      Background
      Inhibition of signaling pathways interfering with cell proliferation and angiogenesis may increase anti-tumor efficacy. Sorafenib as well as mTOR inhibitors showed preliminary activity in KRAS mutated NSCLC.

      Methods
      In the dose escalation part, patients with relapsed solid tumors were treated with escalating doses of everolimus from 2.5-10.0 mg daily p.o. in a 14 days run-in phase followed by the combination with a fixed dose of sorafenib 400 mg bid p.o. The extension phase is currently recruiting patients with KRAS mutated NSCLC. The KRAS mutation status is determined by PCR based high resolution melting curve analysis (HRM) on DNA extracted from FFPE material and validated using Sanger sequencing. HRM has now been replaced by multiplex PCR. Pharmacokinetic (PK) analyses are performed during run-in and during the combination. Treatment outcome is validated with CT scans on day 57.

      Results
      In the dose escalation part, 19 patients were recruited. The dose limiting toxicity (DLT) was not reached. At everolimus dose level of 10 mg/day, increased rates of grade 3 thrombocytopenia (3 patients), leukocytopenia (2 patients) and anaemia (2 patients) occurred after the DLT interval of 29 days. Based on these observations, the dose level of 7.5 mg/day everolimus in combination with 400 mg sorafenib bid was defined as a maximal tolerated dose. The AUC and Cmax values of everolimus at all dose levels were comparable on days 5 and 14. On day 29, AUC and Cmax of everolimus showed a 20 - 40% reduction when co-administered with sorafenib. The best treatment outcome on day 57 was stable disease in 11 patients. Median PFS and OS were 3.7 and 5.5 months, respectively. The extension phase in KRAS mutated NSCLC is currently ongoing. Nine patients have been recruited so far. The CT response at day 57 compared to the baseline of four evaluable patients is ranging from -22% to +5% in the sum of the longest diameter of all targeted lesions.

      Conclusion
      Treatment of patients with relapsed solid tumors with the combination of 7.5 mg everolimus p.o. daily and 400 mg sorafenib p.o. bid is safe and feasible. Current results of an extension phase in KRAS mutated NSCLC patients show preliminary clinical activity in this patient group with an unfavorable prognosis.

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      P1.11-043 - BARIS: A phase I trial to evaluate the safety and tolerability of combined BIBF 1120 and RAD001 in solid tumors and to determine the maximum tolerated dose (MTD) of the combination. (ID 3222)

      09:30 - 09:30  |  Author(s): M. Gardizi

      • Abstract

      Background
      Simultaneious inhibition of several signalling pathways involved in angiogenesis as well as in tumor cell growth regulation by kinase inhibitor combination therapy may increase therapeutic efficacy. Here we evaluate the combination of the mTOR-inhibitor RAD001 (everolimus) and the triple kinase (FGFR, VEGFR, PDGFR) inhibitor BIBF 1120 in a phase I trial in advanced solid tumors. In addition we use DCE-MRI for early identification of patients with benefit from BIBF 1120.

      Methods
      This is an open-label, monocentric phase I trial with 3 dosage arms in a classical „3+3“-design: Patients in arm A receive 5 mg of RAD001 and 2 x 150 mg BIBF 1120, in arm B 10 mg RAD001 and 2 x 150 mg BIBF 1120 will be administered, whereas in arm C, 10 mg of RAD001 and 2 x 200 mg BIBF 1120 will be given. There is no interindividual dose escalation, and the enrollment of the patients will be performed sequentially. Eligible are all patients with relapsed or refractory advanced/metastatic solid tumors (UICC stage IV) and an ECOG performance state of 0-1 for whom no further standard therapies are available and who have predefined adequate organ functions. All patients will start with a 2-week run-in phase of 2 x 200 mg BIBF 1120. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) scans will be performed at baseline staging, on day 3 and day 14. On day 14, there will also be 12 hours-pharmacokinetic (PK) assessment. Combination therapy within the forementioned dosage arms starts on day 15. After two weeks of combination therapy, on day 29, a DCE-MRI scan and 12-hours PK will be performed. Restaging for the evaluation of the efficacy will be performed on day 57. The safety of this combination will be assessed throughout the complete therapy phase using CTC-AE V4.0, with predefined dose-limiting toxicities (DLTs) being assessed until day 42. Patients who experience clinical benefit (i. e., response or stable disease) on day 57 with adequate tolerability of the combination will further receive the medication, as long as the benefit lasts.

      Results
      10 patients have been enrolled so far. In one patient with FGFR-amplified lung cancer, there was a partial response after six weeks of therapy. No DLTs were detected within the first dosage step. Tolerability of the combination was good, as there were no toxicities of CTC-AE grade 3 or greater. In arm B, there has been one DLT (elevation of transaminases), which turned out to be reversibel.

      Conclusion
      So far, the combination of BIBF 1120 and RAD001 seems tob e very good tolerated, demonstrating activity in a patient with NSCLC and FGFR1-amplification. Enrollment into the second dosage stage has already started. We expect the termination of the trial by winter 2013/2014.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P2.11-045 - TRY: A phase II study to evaluate safety and efficacy of combined trastuzumab and AUY922 in advanced non-small-cell lung cancer (NSCLC) with HER2 overexpression or amplification or mutation. (ID 3057)

      09:30 - 09:30  |  Author(s): M. Gardizi

      • Abstract

      Background
      HER2 amplifications and/or mutations are rare genetic alterations in NSCLC accounting for approximately 4%. Preliminary clinical data suggested efficacy of trastuzumab in patients with HER2 IHC3+ status or FISH positivity. The heat shock protein HSP90 is a molecular chaperone that modulates stability and/or transport of intracellular client proteins including HER2. In breast cancer HSP90 inhibition has shown anticancer activity in HER2-positive patients after trastuzumab failure. Here we are investigating the efficacy of the combination of trastuzumab and the HSP90 inhibitor AUY922 in lung cancer patients with aberrant HER2.

      Methods
      This phase II study recruits metastatic NSCLC patients with HER2 overexpression (immunohistochemistry, DAKO-score 3+) or amplification (fluorescence in situ hybridization) or activating mutation after at least one previous standard treatment. In the first part of the study, patients are treated with trastuzumab only. CT scans are scheduled every 6 weeks during treatment. In case of disease progression, patients receive the combination of trastuzumab and AUY922.

      Results
      The study was initiated this year and NSCLC patients are screened within the Network of Genomic Medicine Lung Cancer on HER2 overexpression, amplifications and mutations. Until now, we tested 720 tumor samples by FISH and 63 by genomic sequencing. We identified 55 patients with HER2 amplification, 34 with HER2 overexpression (Dako score 3+) and 7 patients showed a mutation in the HER2 gene (1 exon 19; 6 exon 20).

      Conclusion
      HER2 overexpression, amplification or mutation is a rare genetic alteration in NSCLC patients. Data on treatment with HER2 antibody trastuzumab and HSP90 inhibitor AUY922 will be presented.