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H.T. Tran



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    MO15 - Novel Genes and Pathways (ID 89)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO15.08 - KDR (VEGFR-2) copy number gains and mutations are targetable alterations in non-small cell lung cancer (ID 1466)

      17:00 - 17:05  |  Author(s): H.T. Tran

      • Abstract
      • Presentation
      • Slides

      Background
      Therapeutic regimens targeting the vascular endothelial growth factor (VEGF) pathway have been extensively tested in the treatment of malignancies including non-small cell lung cancer (NSCLC). VEGF pathway inhibitors including bevacizumab or VEGF receptor (VEGFR) tyrosine kinase inhibitors (TKIs) have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, occurring only in subsets of patients. Therefore, predictive markers to identify patients likely to derive benefit are critically needed. Although expression of VEGFR-2, also known as KDR, was initially thought to localize primarily on endothelial cells, VEGFR-2 has been detected on malignant cells. We recently observed that KDR copy number gains (CNGs) were detectable by FISH in ~30% of both adenocarcinoma and squamous cell carcinoma and were associated with poor clinical outcome in early stage NSCLC patients treated with adjuvant chemotherapy. In addition to CNGs, mutations and polymorphisms within the KDR gene were also observed. The impact of these alterations is unknown. Here, we investigated KDR CNGs, polymorphisms, and mutations in NSCLC and their effects on sensitivity to VEGFR targeting agents in preclinical models and in NSCLC patients.

      Methods
      Cell migration was evaluated by Boyden chamber assay. NSCLC cell lines were treated with VEGF pathway inhibitors for 24 hours, and protein lysates where collected. HIF-1α levels were evaluated by ELISA assay. VEGFR, p38, and p70s6K were evaluated by Western blotting. Tumor DNA and peripheral blood DNA, were analyzed in duplicate using Affymetrix Genome-Wide SNP Array 6.0. Transformation of Ba/F3 cells was evaluated by an IL-3-independent growth assay.

      Results
      In tumor cells with KDR CNG, VEGF stimulation induced activation of p38 and p70S6K, and VEGFR TKIs including sorafenib and vandetanib effectively inhibited VEGF-mediated signal transduction. In tumor cell lines with KDR CNG, exogenous VEGF ligand increased cell motility and this was inhibited by VEGFR blockade with TKIs including sunitinib, sorafenib, and axitinib. Various receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxia compared to NSCLC cell lines without KDR CNG. In NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG had a partial response to the VEGFR inhibitor, sorafenib. In addition to gene amplification, mutations and polymorphisms within the KDR gene were also observed. KDR mutation 1586A>T and polymorphism 1416A>T effectively transformed Ba/F3 cells. Finally, we report two clinical cases in which NSCLC patients with the 1416A>T polymorphism had a partial response the VEGF pathway inhibitor, bevacizumab.

      Conclusion
      Collectively, our data indicate that KDR amplification promotes downstream signaling events including activation of the p38, mTOR, and HIF pathways and are targetable by VEGF pathway inhibitors. KDR gene alterations may be predictive markers for VEGF pathway inhibitors.

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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.02 - Proteomic analysis identifies baseline PI3K/Akt pathway activation and treatment-induced supppression of mTOR signaling as determinants of response to MEK inhibition (ID 2845)

      10:35 - 10:40  |  Author(s): H.T. Tran

      • Abstract
      • Presentation
      • Slides

      Background
      Inhibition of MEK is a promising treatment strategy for non-small cell lung cancer (NSCLC). MEK inhibitors are being investigated for KRAS mutant disease, but KRAS alone is not predictive of efficacy, and other predictors of response and resistance are not known. The downstream effects of MEK inhibition have not been fully described. Here, we report broad proteomic analysis of NSCLC cell lines before and after treatment with MEK inhibitor BAY86-9766.

      Methods
      We treated 109 NSCLC cell lines with BAY86-9766. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3[rd] of those tested. Proteomic analysis for regular and phospho-proteins was performed by reverse phase protein array. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines.

      Results
      Increased activation of the PI3 kinase pathway at baseline correlated with resistance to MEK inhibition, with resistant cell lines showing higher baseline levels of pAkt (S437), pAkt (T308), pPDK1, and p4E-BP1 (S65), and lower baseline levels of PTEN (all p<0.05). Cell lines with increased MEK phosphorylation at baseline were more sensitive to MEK inhibition (p=0.048). BAY86-9766 was very effective at reducing pERK (p=1.65x10[-35]) but this modulation was not significantly different between sensitive and resistant cell lines (p=0.64). Increased phosphorylation of MEK was seen with treatment (1.66x10[-16]). mTOR signaling was suppressed by MEK inhibition, with decreased phospho-p70S6K, pS6 (S235/236), and pS6 (S240/S244) and increased eIF4E following treatment (all p<0.02). These effects were significantly more pronounced in sensitive vs resistant cell lines (all p<0.01). Higher levels of LKB1 total protein, pAMPK, and pTSC2 were also seen following treatment (all p<0.02).

      Conclusion
      We have performed broad proteomic analysis of NSCLC cell lines treated with MEK inhibitor BAY86-9766. Baseline activation of the PI3K/Akt pathway predicts for resistance to MEK inhibition. Sensitive cell lines, but not resistant cell lines, show suppression of mTOR activity with treatment with BAY86-9766. The effects of MEK inhibition of mTOR may be modulated by p90RSK through an LKB1 dependent pathway. This suggests a basis for combining targeted agents to overcome resistance, such as combinations of MEK inhibitors with PI3K inhibitors or mTOR inhibitors.

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    P2.06 - Poster Session 2 - Prognostic and Predictive Biomarkers (ID 165)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.06-040 - Circulating cytokines and angiogenic factors (CAFs) as markers of clinical response in a randomized phase II study of trametinib vs docetaxel for patients with KRAS-mutant non-small cell lung cancer (NSCLC) (MEK114653, NCT01362296) (ID 2880)

      09:30 - 09:30  |  Author(s): H.T. Tran

      • Abstract

      Background
      Trametinib is a reversible and highly selective allosteric inhibitor of MEK1/MEK2. In a 2:1 randomized, open-label phase 2 study, trametinib showed an overall response rate of 12% but did not show improvement in progression-free survival (PFS) over docetaxel as second-line treatment in patients with KRAS-mutant NSCLC. Median overall survival (OS) was 8 months in the trametinib arm and has not been reached in the docetaxel arm (ASCO 2013 abstract #8029). Several CAF markers were identified as prognostic and predictive of clinical benefit in patients with renal cell carcinoma (Tran, Lancet 2012), and of tumor shrinkage in patients with NSCLC (Nikolinakos, Cancer Research 2010), after receiving targeted therapy.

      Methods
      Of 134 patients randomized, plasma samples (n = 116 baseline [113 KRAS-mutant], n = 89 day 22) from patients who consented for this optional study were analyzed for 38 CAFs using SearchLight multiplex assays in a CLIA-certified laboratory. Baseline CAF levels were tested for association with PFS using proportional hazards regression within and between arms. Correlation between baseline CAFs and baseline tumor burden was assessed using the Spearman rank correlation test. Change from baseline was assessed using Wilcoxon signed rank tests. P < .01 was considered significant; for treatment arm by CAF-level interaction, P < 0.05 was considered significant.

      Results
      Lower baseline levels of IL-6 and OPN and higher baseline levels of TRAIL were associated with longer PFS in patients treated with trametinib but not those treated with docetaxel. Interaction between CAFs and trametinib or docetaxel treatment was significant for IGFBP-1, MMP-9, E-selectin, and VEGF. There was a positive correlation between IL-6 levels and baseline tumor burden. At day 22, decreases (trametinib: ANG-2, IGF-1, IGFBP-3, IL-10, IL-2R, TIMP-1; docetaxel: IL-6, MIP-1A, MIP-1B, SDF-1) and increases (trametinib: IGFBP-1, IL-6, MMP-2, PIGF, VEGF) in CAF levels were observed. Additional results (pairwise correlation, CAF levels and OS, change from baseline CAF levels and PFS and OS) will be reported.

      Conclusion
      Circulating baseline CAF levels may be predictive of PFS for patients treated with trametinib or docetaxel. CAF levels are modulated by each treatment. The impact of circulating baseline CAFs on PFS warrants additional investigation in well-designed clinical trials. Plasma CAF profiling may aid in the prognostic evaluation of patients and determine potential therapeutic response to trametinib treatment in patients with NSCLC.