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N. Hirsch



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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.01-008 - Inhibition of Binding at Exon 4 of Osteopontin Increases Apoptosis and Apoptotic Protein Expression in Non-Small Cell Lung Cancer (ID 2885)

      09:30 - 16:30  |  Author(s): N. Hirsch

      • Abstract

      Background
      Osteopontin (OPN) is a ubiquitous extracellular protein associated with a wide range of normal and pathologic functions. It is a central regulator of the malignant phenotype in non-small cell lung cancer (NSCLC) through binding of cell surface receptors, but underlying mechanisms are poorly understood. Among OPN’s critical roles in NSCLC progression, is preventing apoptosis related to oxidative stress by activating alternative survival pathways. While OPN’s central RGD domain is considered important to this function, we hypothesize that exon 4, in the amino terminus, which is present in OPN’s A and B isoforms, but not in the C isoform, is essential to this process. We sought to determine the impact of inibition of binding between NSCLC cells and OPN exon 4 on apoptosis and apoptotic protein expression in NSCLC.

      Methods
      A 16 amino acid peptide mimicking the central sequence of OPN exon 4 and a scrambled sham were constructed. Competitive binding assays had previously determined that the OPN exon 4 peptide binds the NSCLC cell surface and inhibits OPN binding. Two NSCLC cell lines with wt p53, A549 (moderate endogenous OPN) and H460 (high endogenous OPN), were placed in serum-free media with OPN exon 4 peptide or scrambled peptide for 48 hours. Cells were then evaluated by TUNEL assay or harvested, lysed and protein expression measured using Human Apoptosis Array (R&D, Minneapolis, MN).

      Results
      Exon 4 peptide treatment resulted in significant increases in apoptosis in both cell lines (Fig). A similar pattern of change in apoptotic protein expression was seen in both cell lines, with significant increases in Bax, cleaved Caspase-3, CytC, Hsp32, Pon2, Cdnk1, and p53-pS15, p53-pS46, and p53-pS392, while Survivin and Claspin expression were significantly decreased (Fig). Notably no significant change was seen in Bclx, Bcl2, and pro-Caspase 3 expression. Scrambled peptide had no effect on apoptosis or protein expression. Figure 1

      Conclusion
      Inhibition of binding between OPN exon 4 and NSCLC cells significantly increased the rate of apoptosis and expression of proteins which modulate apoptosis associated with oxidative stress, including several key phosphorylated p53 variants. These data implicate OPN exon 4 interactions in NSCLC progression and resistance mechanisms and may explain the importance of OPN’s A and B isoforms as opposed to isoform C in NSCLC pathogenesis.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-018 - Cumalative Biomarker Model Predicts 3-Year Recurrence in Resected Stage I Adenocarcinoma of the Lung (ID 1699)

      09:30 - 16:30  |  Author(s): N. Hirsch

      • Abstract

      Background
      Stage I adenocarcinoma is the most curable form of non-small cell lung cancer (NSCLC), yet recurrence following complete resection is >30%. To improve this, it is important to identify indicators of tumor biology, which can predict a more aggressive disease course so adjuvant therapies can be considered. Osteopontin (OPN) is a regulator of malignant function in NSCLC. In more advanced NSCLC patients, plasma OPN levels correlate with prognosis. We hypothesize that pre-operative plasma OPN in combination with clinical factors can predict recurrence following resection in stage I adenocarcinoma.

      Methods
      A cohort of completely resected stage I adenocarcinoma patients without adjuvant or neoadjuvant therapy was prospectively collected and followed through 3 years. Pretreatment demographics, operative variables, pathologic characteristics, and time to progression were recorded. Histology was classified as solid or mixed with noninvasive features. Pre-operative plasma OPN was measured blinded and in duplicate by ELISA (R&D, Minneapolis, MN) and is reported in ng/ml. Cut points to predict recurrence were determined by X-tile (Yale University, CT) plots.

      Results
      There were 141 patients (50M/91F), 103 were stage IA. Median follow-up was 43.9 months and was complete in all to 3 years. Thirty-nine patients (27.8%) recurred by 3 years. The median pre-operative OPN was 54.9 (range, 2.3 – 150.6). OPN levels correlated with tumor size (r=0.25, p=0.003), but not with age, pack years, t-stage, extent of resection, or invasive histologic component. Median OPN was higher in males than females (62.9 vs. 50.5, p=0.009), and in current smokers compared to former/never smokers (68.5 vs. 53.3, p=0.04). In Cox regression analysis, an increased risk for recurrence was associated with preoperative plasma OPN >49.6 (HR=3.8, CI:1.7-7.8, p=0.001), solid histology (HR=2.5, CI:1.3-4.9, p=0.008) and male gender (HR=2.5, CI:1.3-4.6, p=0.005), but not with size, stage, age, pack years, and extent of resection. A model incorporating preoperative OPN and invasive histologic component stratified recurrence risk for both genders, but was highly significant in females (p=0.006) (Fig). Receiver operator curve (ROC) incorporating sex, OPN and invasive histologic component had AUC=0.76 (CI: 0.6-0.8, p=0.03). Figure 1

      Conclusion
      Circulating OPN provides a view of the tumor micro-environment and can serve as an important indicator of the course of the disease in resected NSCLC. When combined with sex and measures of histologic invasive component, plasma OPN >49.6 form a highly predictive cumulative model to predict early recurrence in resected stage I adenocarcinomas and should be validated to assess its value in selecting patients for adjuvant and tumor prevention protocols.