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P. Bunn



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    MO01 - Lung Cancer Biology - Techniques and Platforms (ID 90)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO01.03 - Transitioning To Next Gen Testing Of Lung Carcinoma (ID 2891)

      10:35 - 10:40  |  Author(s): P. Bunn

      • Abstract
      • Slides

      Background
      The feasibility of multigene testing in a clinical setting has been demonstrated by the Lung Cancer Mutation Consortium (LCMC) which has evaluated over 1000 cases from multiple institutions in a CLIA environment. The initial platforms used by the LCMC were SNaPshot and Ion Torrent, allele specific tests. More recently the sequencing by synthesis method (Illumina) used for whole genome sequencing has been scaled for sequencing of a limited number of targeted genes. In this study we compare the performance characteristics of Next Generation testing on the MiSeq platform with the older allele specific SNaPshot platform and evaluate the applicability of Miseq-based testing to a clinical, CLIA regulated setting.

      Methods
      Two Illumina kits, the TruSeq and TruSight evaluating 221 hotspots in 48 gene and 175 exons in 26 genes, respectively, were compared. To assess analytical sensitivity, cell lines with known mutations and SNPs were titered into liver DNA known to be wild-type for the selected mutations, at tumor cell concentrations ranging from 3% to 50%. In addition, 24 formalin-fixed paraffin-embedded lung tumors that had previously been evaluated by SNaPshot or direct sequencing were tested to compare sensitivities and specificities of methods. Paraffin embedded human tumor tissue samples were enriched for tumor cells by coring of paraffin block or macrodissection using a pneumatic cell collector. DNA was extracted by proteinase K digestion and column chromatography, end repaired and phosphorylated. Libraries were prepared from each sample by ligating index adapters that allow for mixing of samples and binding adapters that link DNA fragments to flow cell. Combined libraries were added to flow cells at an appropriate concentration, clusters generated, and sequencing reaction commenced. Results were evaluated by software developed by Illumina or locally at the University of Colorado.

      Results
      Spiking studies indicated that analytic sensitivity for Miseq at loading quantities of 100 to 300 ng (TruSeq) was ~5% for known KRAS and TP53 mutations and several synonymous polymorphisms in other covered genes, comparable to SNaPshot. For clinical samples, average depth of coverage was 5700 (+/- 2267). Unfiltered results using Illumina software supplied with the Miseq instrument showed an average of 88 heterozygous SNPs, 12 insertions and 17 deletions (uncurated for relevance). All of the mutations that were previously found by SNaPshot were also detected by Miseq TruSeq and TruSight protocols (100% concordance). Variants representing known polymorphisms, synonymous changes and variants identified in the context of low coverage were excluded. Data analysis using locally developed software indicated the presence of 1-9 SNPs in each sample that were not predicted by SNaPshot testing, attributable to the wider coverage of the Miseq platforms. None of the additional mutations represented treatable targets with currently available drugs.

      Conclusion
      Next-generation testing is feasible in a CLIA environment using the Miseq platform. However, rigorous software validation is necessary before this platform can be adopted by a busy clinical laboratory. Software limitations currently being addressed include long turnaround time, inadequate vetting of new and recurrent SNPs for clinical significance and limited software development resources.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P3.05-014 - Eribulin, a tublin targeted chemotherapeutic agent, inhibits the in vitro growth of small cell lung cancer cell lines. (ID 2304)

      09:30 - 09:30  |  Author(s): P. Bunn

      • Abstract

      Background
      Background: New therapeutic strategies are urgently needed for small cell lung cancer (SCLC) which accounts for approximately 29,000 cases annually in the U.S. SCLC tumors have rapid doubling times and a propensity for early development of widespread metastatic disease. There have been no therapeutic advances in recent decades. The microtubule-targeting agent eribulin is a mechanistically-unique inhibitor of microtubule dynamics. Eribulin binds with high affinity to a maximum of 15 distinct beta-tubulin binding sites inhibiting microtubule growth by depolymerization and sequestration of tubulin into non-productive aggregates. Eribulin is currently FDA approved for the treatment of metastatic breast cancer patients with a demonstrated significant increase in overall survival in patients that are refractory or resistant to multiple chemotherapy agents. We investigated the effects of eribulin in a panel of 15-human SCLC lines.

      Methods
      Methods: Growth inhibition by eribulin was assessed by tetrazolium based assays at 5-days post treatment with varying drug concentrations and the growth inhibitory (GI50) was calculated. Erbulin induced cell cycle arrest was monitored following propidium iodine staining and analysis by FACS. Apoptosis was determined by using the DNA binding dyes YOPRO and PI and analysis by FACS.

      Results
      Results: Eribulin inhibited the growth of 13 of the cell lines. Four SCLC lines had GI50s of < 1 nM and 9-lines had eribulin GI50 values of 1-6 nM. These GI50 are similar to those reported in breast cancer cell lines. The eribulin IC50 value in the remaining 2-lines was > 100 nM. We are currently exploring clinical and gene expression differences that may explain the high sensitivity and resistance of different cell lines. A two to three-fold increase in the % cells in the G2/M phase of the cell cycle were observed following an 18-hour exposure to eribulin at concentrations of ≤ 5 nM in all cell lines growth inhibited by eribulin. Apoptosis assays are ongoing and in vitro studies of eribulin in combination with radiation are planned.

      Conclusion
      Conclusions: Erbulin inhibited the growth of SCLC lines and induced a significant G2/M arrest. Confirmation of growth inhibition of SCLC cell lines in an in vivo nude mouse model would support human studies in SCLC patients.

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    PL03 - Presidential Symposium Including Top Rated Abstracts (ID 85)

    • Event: WCLC 2013
    • Type: Plenary Session
    • Track:
    • Presentations: 1
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      PL03.07 - Treatment with Therapies Matched to Oncogenic Drivers Improves Survival in Patients with Lung Cancers: Results from The Lung Cancer Mutation Consortium (LCMC) (ID 2444)

      09:21 - 09:33  |  Author(s): P. Bunn

      • Abstract
      • Slides

      Background
      Detecting and targeting the oncogenic drivers EGFR and ALK have transformed the care of patients with lung adenocarcinomas. The LCMC was established to use multiplexed assays to test tumors for alterations in 10 genes and provide the results to clinicians to select treatments and clinical trials matched to the driver detected.

      Methods
      Fourteen LCMC sites enrolled patients with metastatic lung adenocarcinomas and tested their tumors in CLIA laboratories for activating mutations in 10 oncogenic driver genes.

      Results
      Tumors were tested from 1,007 patients for at least one gene and 733 for all 10 genes. An oncogenic driver was found in 466 (64%) of fully-genotyped cases. Among these 733 tumors, drivers found were: KRAS 182 (25%), sensitizing EGFR 122 (17%), ALK rearrangements 57 (8%), “other” EGFR 29 (4%), two genes 24 (3%), HER2 19 (3%), BRAF 16 (2%), PIK3CA 6 (1%), MET amplification 5 (1%), NRAS 5 (1%), MEK1 1 (<1%), AKT1 0. For cases with any genotyping, we used results to select a targeted therapy or trial in 275 (28%). Among 938 patients with follow-up, the median survivals were 3.5 years for the 264 with an oncogenic driver treated with genotype-directed therapy, 2.4 years for the 318 with an oncogenic driver with no genotype-directed therapy, and 2.1 years for the 360 with no driver identified (p<0.0001).

      Conclusion
      Individuals with lung cancers with oncogenic drivers receiving a corresponding targeted agent lived longer than similar patients who did not. An actionable driver was detected in 64% of tumors from patients with lung adenocarcinomas; more than one was present in 3%. Multiplexed testing aided physicians in choosing therapies and targeted trials in 28% of patients. This paradigm for care and research will expand as genotyping becomes more efficient with Next-Gen platforms, additional drivers are identified (i.e.ROS1 and RET), and more targeted drugs become available in the pharmacy and through clinical trials. Supported by HSS NIH NCI 1RC2CA148394-01. Trial Registered with Clinicaltrials.gov: NCT01014286.

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