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P.A. Bunn



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    MO05 - Prognostic and Predictive Biomarkers II (ID 95)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO05.02 - Overexpression of FGFR1 mRNA and protein are more frequent than FGFR1 gene amplification in non-small cell lung cancer (NSCLC) patients (ID 2459)

      16:20 - 16:25  |  Author(s): P.A. Bunn

      • Abstract
      • Presentation
      • Slides

      Background
      Somatic mutations and gene fusions have been identified as oncogenic drivers in lung cancer, however, a number of lung cancers have no apparent molecular aberration driving oncogenesis. It appears that gene/protein overexpression may sustain these “pan-negative” cancers. Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate cell proliferation, differentiation, migration and survival and dysregulation of this signaling pathway is observed in a proportion of lung cancers. A number of compounds targeting FGF/FGFR are in clinical development but clinically applicable biomarker assays and companion diagnostics that accurately identify patients with tumors sensitive to these agents are needed. We previously presented cell line data demonstrating that FGFR1 mRNA (ME) or protein expression (PE) better identified FGFR1 inhibitor sensitive tumors compared to gene copy number (GCN). The goal of this study was to examine FGFR1 ME, PE and GCN in a surgically treated NSCLC clinical cohort and explore possible associations with clinical features and prognosis.

      Methods
      Immunohistochemistry, brightfield in situ hybridization, and silver in situ hybridization were used to investigate ME, PE and GCN, respectively, in a cohort of 189 NSCLC surgically-treated patients. PE was scored by the H-score method (0-300) and ME on a semiquantative integer scale (0-4+), both evaluating the entire tumor specimen. GCN was scored on continuous scale by counting the individual signals in 50 cells and determining the average GCN per tumor cell.

      Results
      Amplification (GCN >=4) was present in 8% of the entire cohort and in 11% of the squamous cell carcinoma (SCC) or mixed histology subgroup. No amplifications were found in the adenocarcinomas (ADC) or tumors from never smokers. In contrast, 29% of SCC and ADC patients had high ME (= 4+). Elevated PE (>= 100) was observed in 20% of the cohort, with the highest expression observed in SCC/mixed histology, but 6% of ADCs also showed elevated PE. There was no elevated FGFR1 PE in the never smokers. There was significant correlation but incomplete overlap between biomarkers. There were no prognostic associations, either with overall or disease-free survival, for FGFR1 GCN, ME, or PE. There was excellent inter-observer agreement among the readers of all 3 biomarker assays.

      Conclusion
      Overexpression of FGFR1 mRNA and protein are more frequent than FGFR1 gene amplification in NSCLC patients. Although GCN amplification was restricted to SCC, elevated ME and PE were found in both ADC and SCC. There was no prognostic association with FGFR1 GCN, ME, or PE. These data are consistent with our previous cell line data that showed elevated PE and ME in non-amplified cells and suggests that GCN may not identify all the potential patients who could benefit from FGF/FGFR pathway inhibitors.

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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): P.A. Bunn

      • 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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    P2.05 - Poster Session 2 - Preclinical Models of Therapeutics/Imaging (ID 158)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.05-017 - Evaluation of the selective aurora B kinase inhibitor AZD1152 in SCLC lines with and without MYC family amplification. (ID 2877)

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

      • Abstract

      Background
      Background: Small cell lung cancer (SCLC) has an overall 5-year survival rate of < 5% despite initial response rates to first line chemotherapy of 70-90%. MYC family amplification occurs in 30% of SCLC and MYC amplified tumors have been shown to be dependent on aurora kinase B for their survival. Aurora B kinase (AURKB) is a chromosomal passenger protein that plays a critical role in mitosis by regulating chromosome alignment, accurate segregation, and cytokinesis during the mitotic stages. We evaluated the effects of the selective aurora B kinase inhibitor AZD1152-HQPA (active drug) in a panel of 15-SCLC lines.

      Methods
      Methods: Nine lines had MYC-family amplification (MYC-5 lines, MYCL1-2 lines, MYCN-2 lines) and 6-lines had no MYC-family amplification. Growth inhibition by AZD1152-HQPA was assessed by tetrazolium based assays. Apoptosis was determined using the DNA binding dyes YOPRO and propidium iodine (PI) and analysis by flow cytomery. The induction of polyploidy was evaluated by flow cytometry following PI staining of the DNA. The effect of AZD1152-HQPA on anchorage independent growth was determined by soft agar colony forming assays. Finally, we evaluated the in vivo efficacy of AZD1152 (prodrug) on SCLC xenografts in nude mice.

      Results
      Results: AZD1152-HQPA GI50 values for the 4 most sensitive SCLC lines were 11-30 nM and < 25% of untreated control growth was observed at 60 nM. Five lines had GI50 values of 30-60 nM but at 600 nM AZD1152-HQPA cell viability remained at 49-55% of untreated control growth. In the remaining 6-lines cell viability at 600 nM was 60-93% of control growth. Growth inhibition marginally correlated with MYC amplification (p=0.044) and was strongly correlated with MYC + MYCL1 + MYCN amplification (p=0.011). Apoptosis (20-28%) was induced by 30 nM AZD1152-HQPA at 24-48 hours in the most sensitive cell lines. Marked increases in DNA ploidy (8N) was observed after 24 hour exposure to AZD1152-HQPA (30nM) in the most sensitive cell lines and at 48 hours in the 5 intermediate lines and in 2 of the resistant lines. In vivo AZD1152 (i.p. 100 mg/kg/M-F for two weeks) induced tumor regression in nude mice implanted with a sensitive cell line. AZD1152 at 50mg/kg/day inhibited tumor growth; however these tumors began growing following treatment cessation. A resistant line was also implanted into nude mice and AZD1152 at 50 and 100 mg/kg/day caused tumor regression. Polyploidy was induced in this cell line at 48 hours post 30 nM AZD1152-HQPA. Anchorage independent growth in this resistant line was also completely inhibited by AZD1152-HQPA 25 nM. The doses of AZD1152-HQPA used in this study are within the range reported to be clinically achievable.

      Conclusion
      Conclusions: AZD1152-HQPA growth inhibition significantly correlated with MYC-family amplification in SCLC lines but some SCLC lines without MYC-family amplification were also inhibited. Additional biomarkers are needed to identify SCLC patients most likely to respond to AZD1152.