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M. Geraci



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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)

      10:30 - 12:00  |  Author(s): M. Geraci

      • 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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      P2.02-014 - Discovery of circulating protein biomarkers of lung dysplasia (ID 2481)

      09:30 - 16:30  |  Author(s): M. Geraci

      • Abstract

      Background
      Endobronchial dysplasia is a premalignant lesion commonly found in current and former smokers. Identifying and treating these lesions before they progress to lung cancer may improve survival. The iloprost chemoprevention trial demonstrated that supplementation with the prostacyclin analog, iloprost, reduced histologic dysplasia in former smokers (Keith et al. Cancer Prev Res. 2011). However, accurate detection of dysplasia requires invasive bronchoscopy to collect multiple endobronchial biopsy samples. This study is the first step in generating blood-based markers of dysplasia to identify individuals at high risk for developing lung cancer and who could benefit from chemoprevention treatment.

      Methods
      Baseline serum samples (n=70) collected from current and former smokers enrolled in the iloprost chemoprevention trial were analyzed with the SOMAscan proteomic platform, which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV. To characterize dysplasia, 6 standardized endobronchial sites, as well as any others that appeared suspicious by either white light or autofluorescence visualization, were biopsied from each study participant and scored by expert pathologists. Samples were stratified by worst biopsy score (Max) for proteomic analysis. Biomarkers correlating with Max pathology score were identified using principal component analysis (PCA), a multivariate technique to identify correlated variables, and the univariate, non-parametric Kolmogorov-Smirnov test (KS test). Serum proteins correlating with pulmonary function were also analyzed.

      Results
      Six proteins correlated with the progression of Max pathology. The change in serum level of these proteins ranged from 14-50% when comparing the lowest (n=16) and highest (n=39) Max pathology score groups. The proteins function in neoplastic progression, cell adhesion, inflammation and metabolic regulation. The protein with the most significant change (FDR correct p value = 0.05) regulates plasma clearance of steroid hormones. The serum protein most strongly correlated with lung function in our study was VEGFR2, which mediates VEGF induced endothelial proliferation and is known to be reduced in the lungs of smokers and patients with COPD and emphysema.

      Conclusion
      Our preliminary results of serum biomarkers associated with preneoplastic dysplasia warrant further study. If validated, this serum-based test to identify individuals who may benefit from chemopreventive intervention could impact lung cancer survival. This work was supported by NCI grants CA 58187 and CA165780.