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I.I. Wistuba



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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): I.I. Wistuba

      • 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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    MO19 - Lung Cancer Immunobiology (ID 91)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO19.10 - Prevalence and prognostic association of PD-L1 protein and immune gene expression in NSCLC (ID 2437)

      11:25 - 11:30  |  Author(s): I.I. Wistuba

      • Abstract
      • Presentation
      • Slides

      Background
      Programmed Death Ligand 1 (PD-L1, CD274, B7-H1) is an immune checkpoint molecule that binds to the receptors PD-1 and B7.1 on activated T cells. Binding negatively regulates T-cell function in both physiological and pathological conditions. Recent clinical studies have suggested that numerous cancers, including NSCLC, may utilize PD-L1 expression to escape T-cell mediated cytotoxic activity. Inhibition of PD-L1 can restore anti-tumor immunity, leading to clinical responses. A better understanding of PD-L1 expression patterns, co-expression with other immune markers and actionable disease associated biomarkers may provide insight into the future design of cancer immunotherapy trials in NSCLC.

      Methods
      Expression of PD-L1 was measured by immunohistochemistry (IHC) in archival tumors and, in some cases, in paired metastases in 2 FFPE NSCLC tumor tissue collections. Set 1 (N=561) was collected from patients who were eligible for surgery with curative intent from 2003 to 2005 at MD Anderson Cancer Center. The samples from Set 2 (N=300) contained surgically resected NSCLC tissue collected between 2006 and 2011 (UCCC and Norwegian Radium Hospital). PD-L1 expression was analyzed in both malignant and non-malignant cells (e.g., infiltrating immune cells). In addition, a multiplex qPCR assay that measures ≈90 immune-related genes was used to characterize the tumor immune microenvironment in the NSCLC tumor samples. Disease associated biomarkers, including the mutation status of EGFR and KRAS, as well as expression of MET (by IHC) were also evaluated.

      Results
      Prevalence of PD-L1 was comparable between adenocarcinoma and squamous cell carcinoma (≈30% in tumor cells; ≈45% and ≈50%, respectively, in immune cells). PD-L1 prevalence varied depending on the pathological stage, and was higher in Stages I-IIIA than in Stages IIIB-IV. Similarly, the prognostic value of PD-L1 varied by both stage and histology. In adenocarcinoma, tumors with PD-L1–positive tumor cells had a higher frequency of KRAS mutation and high Met expression, and a lower frequency of EGFR mutation compared with PD-L1–negative tumors. In contrast, tumors with PD-L1–positive and PD-L1–negative immune cells had a comparable frequency of high Met expression. Expression of PD-L1 was frequently co-localized with CD8+ T-cell infiltrates. Gene expression profiling revealed differences in the tumor immune environment, including genes associated with cytotoxic T-cells, between adenocarcinomas and squamous cell carcinomas. PD-L1 protein and immune gene expression associations with patient characteristics will be described in further detail.

      Conclusion
      These data provide a comprehensive description of PD-L1 expression in the context of disease biology utilizing large independent cohorts of well-characterized lung cancer tissues. The results highlight the complexity of the tumor immune environment in NSCLC with particular emphasis on the association with factors such as pathological stage, histology and oncogenic mutational status. These analyses may help guide future development of immunotherapy trials in NSCLC.

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    O04 - Molecular Pathology I (ID 126)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      O04.02 - Using NGS for Mutational Profiling of NSCLC in the Clinical Setting (ID 2571)

      10:40 - 10:50  |  Author(s): I.I. Wistuba

      • Abstract
      • Presentation
      • Slides

      Background
      Recent advances in molecular characterization of lung cancer have led to the identification of potential therapeutic targets that play key roles in regulating cell growth and proliferation. With the introduction of new targeted therapies, it becomes increasingly important to accurately characterize mutation status in lung cancer patients to provide personalized care that define prognosis and predict response to therapy. The advent of next generation sequencing (NGS) platforms in the realm of clinical molecular diagnostics has made multi-gene mutational profiling an affordable and highly successful methodology for massively parallel sequencing using small quantities of DNA.

      Methods
      Tumor specimens from 262 distinct samples of primary lung carcinoma including adenocarcinoma (n=228), squamous cell carcinoma (n=15), non small cell cancer not otherwise specified (NSC-NOS) (n=8), poorly differentiated carcinoma (n=4), neuroendocrine carcinoma (n=2), small cell carcinoma (n=1) and pleomorphic carcinoma (n=4) were tested by NGS. Tumor samples included formalin-fixed paraffin-embedded surgical core needle biopsies, resection specimens, cytopathology cell blocks, as well as cytopathology direct smears. Ten ng of DNA from each sample was tested for mutations in hotspot regions of 46 cancer related genes (Ion AmpliSeq Cancer Panel) using either a 316 chip or a 318 chip on an Ion Torrent Personal Genome Machine (PGM) Sequencer (Life Technologies, CA).

      Results
      Mutations were detected in 222/240 (93%) patients with a histologic diagnosis of adenocarcinoma, NSC-NOS or PDC. EGFR mutations were detected in 47 (20%) of these patients and double EGFR mutations identified in 13 cases, including acquired resistance mutations T790M (n=6) and S768I (n=3). KRAS mutations were detected in 61 (25%) cases, most commonly involving codons 12 and 13 (n= 58) and less frequently involving codons 61 and 146 (n= 3). TP53 was most frequently mutated (n=65; 27%) and was often seen in conjunction with EGFR mutations (n=14; 5%) and KRAS mutations (n=15; 6%). Mutations were detected in 10/15 (67%) squamous cell carcinomas with mutations in TP53 (n=5), CDKN2A (n=3) and PIK3CA (n=2) most frequently seen. Additional mutations detected at a lower frequency from the entire dataset were STK11, ATM, BRAF, PIK3CA, CTNNB1, IDH1, NRAS, CDKN2A, KDR, RET, MET, FBXW7, APC, RB1, FLT3, GNAS, ABL1, HRAS, PTPN11, JAK3, NOTCH1, SMAD4, SMARCB1, SMO, MLH1, AKT1, and ERBB4.

      Conclusion
      In summary, our results show that NGS-based mutational profiling using small amounts of DNA derived from FFPE as well as cytology smears can provide important information regarding mutation status of genes that play key roles in growth and progression of tumor in lung cancer patients and can provide insight into directing personalized cancer therapy.

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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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      P1.01-003 - Targeting EMT in lung cancer: An integrated analysis of Axl and other mesenchymal targets in The Cancer Genome Atlas (TCGA) (ID 1991)

      09:58 - 10:12  |  Author(s): I.I. Wistuba

      • Abstract

      Background
      We previously developed a 76-gene signature of epithelial-to-mesenchymal transition (EMT) that predicted resistance to EGFR and PI3K inhibition in non-small cell lung cancer (NSCLC). This analysis also identified Axl, a receptor tyrosine kinase, as a novel target for mesenchymal lung cancers. Here, we conducted an integrated molecular analysis of EMT in resected, treatment-naïve tumors from three clinical cohorts, including the Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC), with particular focus on Axl as a potential target in mesenchymal NSCLC.

      Methods
      Using our 76-gene EMT signature, TCGA patient tumors (230 LUAD, 178 LUSC) and a large MDACC cohort of resected tumors (n=279) were assigned an “EMT score.” Expression of >160 total and phosphoproteins were measured in the tumors by reverse phase protein array (RPPA). Proteomic profiles and other molecular markers (including mutation status, miRNA expression, and copy number) were correlated with EMT scores and Axl expression levels.

      Results
      The EMT score, derived from our EMT signature, identified NSCLC tumors with mesenchymal gene expression signatures (average 23% of tumors across all cohorts, range 14-34%). In both LUAD and LUSC, EMT scores were highly correlated with (1) expression levels of the miR200 family, a group of miRNAs previously known to regulate EMT (p-values <0.001 by Pearson correlation) and (2) levels of proteins central to EMT (e.g., E-cadherin, alpha-catenin, beta-catenin, claudin-7, fibronectin; p<0.001 for all). Mesenchymal tumors also had lower expression of TTF1 in LUAD (p=0.0002) and lower p63 in LUSC (p=0.003). Although pEGFR levels were higher in epithelial LUAD tumors (p=0.01), the frequency of EGFR mutations was not significantly higher in this group. EMT score was not associated with smoking status. Consistent with our previous findings in cell lines and patients with advanced NSCLC (BATTLE trial), protein expression of the receptor tyrosine kinase Axl was significantly higher in tumors with mesenchymal signatures (high EMT scores) and with low E-cadherin protein expression (p<0.005 for both). The inverse correlation between tumor E-cadherin and Axl expression was confirmed in an independent group of NSCLC cases by immunohistochemistry. Although a small number of Axl mutations were observed (<3% of tumors), few occurred in the kinase domain and their biological significance is unknown. Other potential therapeutic targets expressed at higher levels in mesenchymal lung cancers included PKC-alpha, NFKB, and FGFR1.

      Conclusion
      The EMT gene expression signature performed well in the TCGA LUAD, TCGA LUSC, and MDACC cohorts, correlating strongly with established markers of EMT on other data platforms (miRNA and protein). We observed strong protein expression of the receptor tyrosine kinase Axl (as well as other targets) among mesenchymal tumors, supporting further investigation of AXL as a potential EMT target and into the mechanism of its overexpression in NSCLC.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.05-005 - VEGF signaling inhibition by cediranib enhances the antitumor and anti-metastatic effects of radiation therapy more substantially than chemotherapy in orthotopic lung cancer models (ID 1443)

      09:30 - 09:30  |  Author(s): I.I. Wistuba

      • Abstract

      Background
      The outcome for lung cancer patients remains poor and new therapeutic approaches are urgently needed. Cediranib is an orally available inhibitor of all 3 VEGFR tyrosine kinases. We evaluated the therapeutic efficacy and radiosensitizing effects of cediranib and paclitaxel, alone or in combination, in orthotopic models of human lung adenocarcinoma that mimic clinical patterns of malignant progression.

      Methods
      PC14PE6 or NCI-H441 human lung adenocarcinoma cells (1 x 10[6]) were injected into the left lungs of nude mice. Mice were randomized (8/group) to treatment with vehicle control, cediranib (3 mg/kg/day po), paclitaxel (200 µg/week ip), radiation to the left lung and mediastinum (20 Gy in 5 fractions over 2 weeks), or radiation with cediranib and/or paclitaxel. When controls became moribund, all mice were sacrificed and assessed for lung tumor burden and mediastinal nodal metastasis. Lung tumors and adjacent tissues were analyzed immunohistochemically.

      Results
      All treatments were well tolerated without significant differences in body weight between groups. In both models, cediranib or radiation therapy alone inhibited tumor growth and lymph node metastasis with efficacy superior to paclitaxel. Cediranib markedly enhanced the antitumor and antimetastatic effects of radiation with 99.3% and 92.1% reductions in primary lung tumor volume in the PC14PE6 and NCI-H441 models, respectively, while paclitaxel only modestly improved the effects of radiation therapy. Trimodality therapy resulted in a near-complete suppression of tumor growth and metastasis, with 99.8% and 98.3% reductions in tumor volume compared to control in the PC14PE6 and NCI-H441 models, respectively, without evidence of lymph node metastasis. Immunohistochemical analyses of lung tumors revealed that cediranib inhibited angiogenesis and tumor cell proliferation and increased tumor and endothelial cell apoptosis. The antiangiogenic and apoptotic effects of cediranib were substantially enhanced when combined with radiation and paclitaxel. Cediranib alone or in combination with radiation and/or paclitaxel increased VEGFR2 expression, but VEGF expression was not significantly impacted by treatment. VEGFR2/3 activation was blocked by cediranib alone or in combination therapy.

      PC14PE6 NCI-H441
      Treatment Left Lung Weight (mg) Left Lung Tumor Volume (mm[3]) Mediastinal Lymph Node Metastasis Left Lung Weight (mg) Left Lung Tumor Volume (mm[3]) Mediastinal Lymph Node Metastasis
      Vehicle 710 (490-1210) 753 (254-1089) 7/8 935 (800-1230) 1146 (860-1601) 8/8
      Paclitaxel 200ug/week 545 (150-860) 506 (37-817) 6/8 785 (485-820) 820 (576-1208) 7/8
      Radiation 20Gy/5fractions 220** (50-360) 154* (34-270) 4/8 485** (330-820) 501* (333-879) 6/8
      Cediranib 3mg/kg/day 215* (70-540) 137* (13-316) 4/8 395** (230-570) 414** (261-698) 5/8
      Radiation +Paclitaxel 185** (60-260) 87** (21-268) 2/8 360** (260-650) 327** (236-651) 5/8
      Cediranib + Paclitaxel 125** (60-260) 41** (0-150) 1/8[†] 225** (160-630) 241** (79-651) 4/8[†]
      Radiation + Cediranib 50* (40-60) 0** (0-28) 0/8[†] 120** (70-190) 88** (1-182) 2/8[†]
      Radiation + Cediranib + Paclitaxel 40** (40-60) 0** (0-1) 0/8[†] 100** (60-120) 9** (1-64) 0/8[†]
      Data are presented as medians and ranges or as incidence. [†]p<0.05 versus vehicle (lymph nodes), *p<0.01, **p<0.001 versus vehicle (others)

      Conclusion
      Trimodality therapy with cediranib, paclitaxel, and radiation resulted in the near complete suppression of lung tumor growth and metastasis with markedly enhanced antiangiogenic and apoptotic effects. The radiosensitizing effects of cediranib upon lung tumors and their vasculature was superior to those of paclitaxel with markedly enhanced apoptosis. The combination of cediranib with radiotherapy or chemoradiotherapy is a potentially promising therapy for cancer and our data provides a strong basis for the design of clinical trials in lung adenocarcinoma patients.