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Y. Miller



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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): Y. Miller

      • 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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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O12.02 - Pathway Analysis of Gene Expression Profiles that Distinguish Persistent from Regressive Bronchial Dysplasia Indicate Synergistic Role for Polo-Like Kinase 1 (PLK1) and Epoxide Hydrolase 3 (EPHX3) in Malignant Progression. (ID 3334)

      10:40 - 10:50  |  Author(s): Y. Miller

      • Abstract
      • Presentation
      • Slides

      Background
      160,000 Americans die from lung cancer annually and the prognosis for invasive lung cancer is poor. Prevention of cancer represents an approach with high potential for significant reduction in mortality. Bronchial dysplasia (BD) is a precursor lesion of squamous cell carcinoma (SCC) of the lung, and persistent BDs represent a high risk subset of these lesions. Genomic instability is an important process underlying malignant progression. Gene expression microarray analyses were used to identify potential mediators of genomic instability in persistent BD and study their activity in these high risk lesions. Two genes, PLK1, which abrogates G2-M checkpoint DNA damage repair, and EPHX3, which converts tobacco smoke derived pro-carcinogens to mutagens, were selected for further analysis.

      Methods
      Sixty-three frozen baseline biopsies were classified into persistent/progressive BD, regressive BD , progressive non-dysplasia and stable non-dysplasia groups according to the presence or absence of BD on follow-up biopsies. H&E staining was performed on frozen sections to confirm histology, and RNA was harvested for global gene expression microarray analysis. Intergroup comparisons employed ANOVA statistical analysis with a false discovery rate of 10% to identify differentially expressed genes associated with persistence and gene expression alterations related to baseline histology used Spearman correlation coefficient cutoff of r= +/- 0.5. A pathway analysis (Ingenuity) using the persistence related genelist was performed to identify active pathways associated with persistence of BD. Validational studies were performed by quantitative RT-PCR in cell lines established from persistent and regressive bronchial sites. Inhibitors of persistence associated enzymes were used in tissue culture based assays of cellular proliferation.

      Results
      Gene expression analyses support the unique biological nature of persistent BD. Intergroup comparisons showed significant numbers of differentially expressed genes only in the comparisons of persistent BD with regressive BD (318 genes) or stable non-dysplasia (6254 genes). 831 genes showed differential expression associated with increasing baseline dysplastic grade regardless of outcome. While approximately half of these genes also differentiated persistent from regressive BD, the presence of numerous persistence related genes that are independent of histology further substantiates the unique high risk nature of persistent BD. A pathway analysis revealed “mitotic roles of PLKs” as having the most significant association with persistence. Quantitative RT-PCR using cultures of 8 persistent BD and 6 regressive BD validated increased expression in persistent BD of PLK1 (2.77X, p=0.002) and EPHX3 (2.36X, p=0.081). Using a classification of dysplastic specimens as high or low expressers of PLK1 and/or EPHX3 (high > mean), we found a significant direct relationship with increased level of outcome diagnosis score: low expression of both genes (2.58); high expression of only one gene (3.60); and high expression of both (5.06). The baseline diagnosis did not differ between groups. Culture of the SCC cell line H2009 with EPHX inhibitor revealed a non-significant trend toward decreased proliferation (80.4% vs untreated).

      Conclusion
      Gene expression data confirms the biologically distinct nature of persistent BD. PLK1 and EPHX3 overexpression demonstrate a cooperative effect in respect to increased outcome histology suggesting a potential role for these enzymes in persistence/progression of BD via promotion of genomic instability.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-015 - Proteomic insights with lung cancer tumors based on histopathologic subtypes and genotypes (ID 2467)

      13:28 - 13:45  |  Author(s): Y. Miller

      • Abstract

      Background
      Proteomic analysis of blood and tissue can reveal essential connections between the biochemical pathways altered in malignancy and tools for cancer diagnosis and treatment. The two major histologic subtypes of non-small cell lung cancer (NSCLC), adenocarcinoma (AD) and squamous cell carcinoma (SQ) differ in prognosis and optimal treatment. Targeting molecular pathways that drive malignancy has led to a paradigm shift in the development of specific treatments for patients based on their tumor mutation profile. We have conducted a comparative proteomic analysis of lung tumor histologic and driver mutation subsets to reveal biomarkers that link critical pathways for cell growth and survival to specific tumor phenotypes and genotypes.

      Methods
      We analyzed 68 NSCLC tumor and matched non-tumor tissue lysates (2 ug total protein/sample) with the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV. The study consisted of 49 AD and 19 SQ tumors, 88% of which were Stage I or II. Somatic driver mutations were identified with multiplex PCR (SnapShot genotyping). Pairwise proteomic comparisons of tumor/non-tumor or AD/SQ tissue samples were performed using the Mann-Whitney test. The non-parametric Kruskal-Wallis test was used to discover differences among multiple pairwise driver mutation comparisons. Dependency network analysis was used to explore correlations enriched in tumor tissue vs non-tumor tissue. The statistical significance of the results was adjusted for multiple comparisons using false discovery rate (FDR) correction.

      Results
      Differences between tumor and non-tumor tissue were dominated by inflammatory, apoptotic and cell proliferation proteins. A total of 79 proteins were significantly different between AD and SQ at a 15% FDR. When compared to non-tumor levels, these proteins divided into 3 phenotypes: AD only (9 proteins), SQ only (19 proteins) or Both (51 proteins). Both refers to proteins that are tumor biomarkers in both AD and SQ and the protein levels are different between AD and SQ. The most common pattern was a progression in protein levels from non-tumor to AD to SQ, whether the pattern was higher or lower in tumor tissue. These proteins are members of cell proliferation and inflammatory pathways. This observation is consistent with the SQ only proteins, which are enriched for angiogenesis, cell proliferation and cell adhesion proteins. Driver mutation analysis revealed 5 inflammatory proteins that were higher in KRAS vs EGFR mutations and a TNF-alpha antagonist that was suppressed in EGFR mutants.

      Conclusion
      Unexpected findings that the AD proteome is closer to non-tumor lung tissue than SQ were revealed through broad proteomic profiling. Alteration in cell proliferation and inflammation pathways discovered in this study may lead to new insights in tumor biology and targeted therapeutics. This work was supported by a grant from the LUNGevity Foundation, NCI grant CA 58187 and Cancer Center Support Grant (P30CA046934).