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Background: Tumor cell regulation of the recruitment and activity of diverse inflammatory cells creates an immunosuppressive microenvironment that favors both tumor growth and metastasis. However, the mechanistic basis of how tumor cells trigger intra-tumoral immunosuppression have not been fully defined. Here we show that metastasis-prone lung adenocarcinoma cells suppress the proliferation and activity of intra-tumoral CD8+ T cells through a PD-L1 dependent mechanism and that PD-L1 expression is regulated by microRNA-200 (miR-200), a known mediator of tumor cell EMT. Results: Analysis of patient tumors from The Cancer Genome Atlas (TCGA) (230 adenocarcinomas) and a large MDACC cohort of resected tumors (N>150) demonstrated that tumors with a mesenchymal gene expression signature had repression of miR-200 family expression and higher levels of PD-L1 expression. These findings were confirmed experimentally in human and murine NSCLC cell lines. In vivo experiments with a Kras/p53 mutant mouse model of lung adenocarcinoma showed that decreased miR-200 led to increased expression of PD-L1 levels on tumor cells, causing profound CD8+ T cell suppression. Upon suppression of CD8+ T cell activity tumors acquired a growth advantage and metastatic capability. Confirmation of these results with the LLC model in PD-L1 knockout mice also demonstrated that PD-L1 expression on the tumor cells, rather than other cells in the tumor microenvironment, was critical to defining tumor immunity and metastastic ability. The metastases could be suppressed by genetic and pharmacologic blockade of PD-L1. Conclusion: Our findings demonstrate a novel biological role for miR-200 in cancer cells by simultaneous control of the cell-intrinsic EMT program and the cell-non-autonomous PD-L1-mediated immune evasion. These findings support further investigation into the mechanistic effects of miR-200 on metastasis and the use of specific EMT-related biomarkers to select immunomodulatory therapies. Citation Format: Don L. Gibbons, Limo Chen, Sangeeta Goswami, Young-Ho Ahn, Lauren A. Byers, Lixia Diao, Jing Wang, Angelica Cortez, Jonathon Roybal, James Welsh, Xuejun Zhang, David Dwyer, Xiaohui Yi, Chen Lieping, Ignacio Wistuba, John V. Heymach, Jonathan M. Kurie, F. Xiao-Feng Qin. microRNA-200 regulates tumor cell PD-L1 expression to control lung cancer metastasis. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A21.

Background: The discovery of driver genes such as EGFR, ALK, and ROS1 in non-small cell lung cancer has led to novel, highly active therapies for a subset of patients. However, the majority of lung adenocarcinomas do not have alterations in established driver oncogenes. Here we used an integrated proteomic and genomic analysis of the Cancer Genome Atlas (TCGA) to identify potential therapeutic targets in oncogene-negative lung adenocarcinomas. Methods: Protein expression was measured by reverse phase protein array in 181 TCGA lung adenocarcinoma tumors. Protein levels were correlated with mutational status by t-test for individual mutations and for oncogene-positive versus negative tumors. A false discovery rate of 0.10 (corresponding p-value ≤0.047) was used for these analyses. Results: Expression of 160 total and phospho-proteins were compared between oncogene-positive and negative tumors using reverse phase protein array (RPPA). Oncogene-positive tumors included those with canonical mutations in KRAS, EGFR, BRAF, ROS1, ALK, RET, MAPK1, HRAS, NRAS, AKT1, MET, or ERBB2. For the two most frequent driver oncogenes, KRAS (n=47) and EGFR (n=27), the top markers expressed in mutated tumors were pRaf/pMAPK/pERK (KRAS mutated) and pEGFR (EGFR mutated) (p Conclusion: Potentially targetable or predictive markers, including Chk1/2, Bim, and thymidylate synthase, were expressed at higher levels in oncogene-negative lung adenocarcinomas. These findings support further investigation of these targets and associated biomarkers and could provide a treatment strategy for patients without established driver mutations such as EGFR, ALK, and ROS1. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C117. Citation Format: Lixia Diao, Pan Tong, Jing Wang, You-Hong Fan, Yiling Lu, Wenbin Liu, Carmen Behrens, Ignacio I. Wistuba, John V. Heymach, John N. Weinstein, Gordon B. Mills, Lauren A. Byers. An integrated proteomic analysis of lung adenocarcinomas from The Cancer Genome Atlas (TCGA) reveals potential targets for oncogene-negative tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C117.

Background: Targeted therapy for head and neck squamous cell carcinoma (HNSCC) is currently limited to EGFR inhibition with agents such as cetuximab. We and others have previously demonstrated that epithelial to mesenchymal transition (EMT) is associated with resistance to EGFR inhibitors in HNSCC and lung cancers but greater sensitivity to other targeted drugs, such as AXL inhibitors. Here, we apply a previously published EMT signature to identify novel potential therapeutic targets (PTTs) in mesenchymal HNSCC tumors (MHNTs). Methods: A high-throughput analysis of gene and protein expression in HNSCC tumors from The Cancer Genome Atlas (TCGA) (A, n=113) was performed to identify PTTs in MHNTs. Specifically, the EMT signature score for each tumor was correlated with marker expression levels by Pearson correlation (mRNA, protein) or t-test (mutation, HPV status). PTTs were validated in an independent cohort of high-risk, resected tumors (B, n=105) and HNSCC cell lines (C, n=60). PTTs were defined as druggable targets found on recent HNSCC publications, our in-house targeted sequencing panel of 202 genes and publicly available cancer cell lines drug sensitivity datasets (DSDs). Results: At a false discovery rate of 1% (p0.5) and confirmed in our independent cohort included DDR2, FGFR1, KDR, MMP14, MMP2, PDGFRA, PDGFRB and TGFBR2. Targets that were overexpressed in both tumors and cell lines included NOTCH4 (A and B p Conclusions: An integrated analysis of EMT and expression data from two independent cohorts identifies previously unrecognized, candidate PTTs in mesenchymal HNSCC tumors. Validation of the PTTs at the protein level is ongoing. The approach described may identify patient subsets, beyond mutational status, for whom targeted therapy can be successful. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A131. Citation Format: Milena P. Mak, Lixia Diao, Jing Wang, Patrick KS Ng, You-Hong Fan, Michael D. Story, William N. William, John V. Heymach, John N. Weinstein, Kevin R. Coombes, Lauren A. Byers. Identification of candidate therapeutic targets in mesenchymal head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A131.

6011 Background: Epithelial-mesenchymal transition (EMT) has been associated with EGFR inhibitor resistance in preclinical studies of head and neck squamous cell carcinoma (HNSCC). Recently, we developed an EMT signature that predicts EGFR inhibitor resistance in lung cancer. Using this signature, we explored the association between EMT and drug response in HNSCC, focusing on the tyrosine kinase Axl as a potential therapeutic target. Methods: We conducted an integrated molecular and drug response analysis in HNSCC. A 76-gene EMT signature previously developed and validated in lung cancer was tested in HNSCC cell lines (n>50) and patient tumors from The Cancer Genome Atlas (TCGA) (n=113) and a MDACC cohort (n=105). Reverse phase protein array (RPPA) and proliferation assays were used to measure protein expression and sensitivity to erlotinib and the Axl inhibitors SGI-7079 and TP-0930. Results: The EMT signature identified distinct epithelial and mesenchymal subsets of HNSCC among cell lines and patient tumors. RPPA experiments revealed higher protein levels of the receptor tyrosine kinase Axl, vimentin, and N-cadherin and lower expression of E-cadherin and beta-catenin in mesenchymal HNSCC (p-values 10μM); however, we discovered that mesenchymal HNSCC were highly sensitive to two Axl inhibitors, SGI-7079 and TP-0930 (IC50s ≤1.2μM and 0.2uM, respectively). Conclusions: Our EMT gene expression signature identified discrete epithelial and mesenchymal subgroups of HNSCC. Mesenchymal HNSCC cells expressed higher levels of Axl protein and exhibited sensitivity to Axl inhibition, but resistance to erlotinib. These results highlight differences in drug response between epithelial and mesenchymal cancers and support Axl as a potential therapeutic target and predictive marker of EGFR inhibitor resistance in HNSCC. (Funded in part by 5 P50 CA097007-10)

Background: LKB1 is a tumor suppressor gene that is lost in ∼35% of non-small cell lung cancers (NSCLC), half of which carry concurrent KRAS mutations. When functioning normally, LKB1 plays a critical role in energy sensing and regulates the PI3K/mTOR pathway. We have previously shown that LKB1 loss is associated with greater resistance to certain targeted drugs. Here, we investigate signaling pathways dysregulated in the setting of LKB1 loss, with a particular focus on potential novel therapeutic targets. Methods: Expression of >140 total and phospho-proteins were measured in 109 NSCLC cell lines by reverse phase protein array (RPPA). Differences in protein expression between LKB1 deficient versus intact cell lines were determined by t-test, with correction for multiple testing. IC50s for targeted therapies were determined by MTS assay in cell lines with and without LKB1. For the drug combination assay, nine NSCLC cell lines with and without LKB1 were treated with the IGFR inhibitor OSI-906 plus serial dilutions of the dual mTOR inhibitor OSI-027. Proliferation was measured after 72 hours. Bliss algorithm was used to calculate synergy and fractional inhibition for each plate independently. Further validation was performed using NSCLC cells stably overexpressing LKB1. Results: Loss of LKB1 via mutation or deletion was strongly correlated with LKB1 protein expression (p Conclusions: These studies identify new potential targets in LKB1 deficient NSCLC, which represents a significant portion of lung cancer patients. Specifically, the combination of IGFR and mTOR inhibition demonstrated synergy, supporting further investigation as a therapeutic approach for this molecularly defined subset of NSCLC patients. Citation Format: Lauren Averett Byers, Maria Angelica Cortez, Chao Yang, Jing Wang, Lixia Diao, You Hong Fan, Luc Girard, Adi Gazdar, Ignacio Wistuba, John D. Minna, Matthew O'Connor, Sharon Barr, John V. Heymach. Proteomic profiling of signaling pathways in LKB1 deficient non-small cell lung cancers (NSCLC) identifies novel therapeutic targets including IGF1R pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2403. doi:10.1158/1538-7445.AM2013-2403

Targeted therapies designed to inhibit the vascular endothelial growth factor (VEGF) pathway have been extensively evaluated in the treatment of malignancies including Non-Small Cell Lung Cancer (NSCLC). VEGF pathway inhibitors such as bevacizumab, or the multitargeted receptor tyrosine kinase inhibitors (TKIs) vandetanib and sorafenib, have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, seen only in subsets of patients. Thus, predictive markers for identifying which patients are likely to benefit are critically needed. Although expression of VEGF receptor-2 (VEGFR-2, also known as KDR) was initially thought to primarily occur in endothelial cells, VEGFR-2 has been detected on malignant cells, including lung cancer cells, and in NSCLC, overexpression of VEGFR-2 on tumor cells is associated with a poor clinical outcome. Amplification of KDR has been detected in lung cancer specimens at a relatively high frequency (9% and 32%). The consequences of KDR copy number gains (CNGs) are not yet understood. Recently, we have shown that NSCLC cell lines with KDR copy number gains (CNGs) were associated with in vitro resistance to platinum chemotherapy, and KDR CNG predicted worse overall survival in patients who received platinum adjuvant therapy but not in untreated patients. We investigated the hypothesis that NSCLC tumor cells with KDR CNG display increased sensitivity to VEGFR TKIs compared to tumor cells without KDR CNG. In tumor cell lines with KDR CNG, treatment with exogenous VEGF ligand enhanced cell motility and this was inhibited by VEGFR blockade with TKIs. Multiple receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxic conditions compared to NSCLC cell lines without KDR CNG. Here, we show that 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 as determined by SNP array had a partial response to VEGFR inhibition with sorafenib. Citation Format: Monique B. Nilsson, Tina Cascone, Jayanthi Gudikote, Emily Roarty, Lixia Diao, Andrew Koo, Sumankalai Ramachandran, Erick Riquelme, Hai Tran, Ignacio Wistuba, David P. Carbone, John Heymach. KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 374. doi:10.1158/1538-7445.AM2013-374

Background: Small cell lung cancer (SCLC) is an aggressive malignancy that accounts for 13% of lung cancers in the US and has a 5-year survival rate Methods: Sensitivity of SCLC lines to BMN673 and cisplatin was determined by CellTiter-Glo cell viability assay. Total and phospho-protein expression of 200 markers was measured at baseline and following drug treatment in SCLC cell lines by reverse phase protein array (RPPA). Correlations between baseline protein expression and drug sensitivity were determined by Spearman rank correlation. Modulation of protein expression post treatment was assessed by ANOVA. Results: The novel PARP inhibitor BMN673 had potent in vitro activity in a panel of 12 SCLC cell lines with IC50’s ranging from 1.7-15nM. A higher expression level of several DNA repair proteins was associated with greater BMN673 sensitivity. For example, protein expression levels of ERCC1, DNA PKcs, ATM, FANCD2, and pChk2 were inversely correlated with IC50 values (Rho values -0.93 to -0.81; p≤0.02). In contrast, high baseline expression of phosphorylated Akt (T308) (R=0.91, p=0.005) and pAkt (S473) (R=0.81, p=0.02) were associated with decreased sensitivity to BMN673. We also observed increased phosphorylation of mTOR, Akt, and S6 (p Conclusions: Here we demonstrate significant, single-agent in vitro activity of the PARP inhibitor BMN673 in SCLC and potential predictive markers of response. Interestingly, for the first time we show an inverse correlation between PARP inhibitor sensitivity and PI3K/Akt/mTOR pathway activation. This observation parallels recent data suggesting that PI3K inhibition may sensitize breast cancer to PARP inhibition and suggests a potential coordinated regulation between DNA repair and PI3K pathways. The activity of BMN673 and these candidate response biomarkers will be further investigated in a Phase I cohort expansion of BMN673 in patients with SCLC. Citation Format: Robert J. Cardnell, Ying Feng, Lixia Diao, You Hong Fan, Jing Wang, Yuqiao Shin, John D. Minna, John V. Heymach, Lauren A. Byers. Proteomic profiling identifies PI3K and DNA repair pathways as potential markers of response to PARP inhibitor BMN673 in SCLC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2489. doi:10.1158/1538-7445.AM2013-2489

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases and 350,000 cancer deaths each year worldwide. It is a disabling and disfiguring cancer with a profound impact eating, speaking, and appearance. We need better systemic treatment for HNSCC. Targeted therapy can be remarkably effective if it inhibits key oncogenic drivers but no such targets have yet been identified in HNSCC. To identify potential targets, we screened sixty validated HNSCC cell lines using Sequenom for a panel of sixty potentially targetable mutations commonly found in squamous cancers. In addition, we used SNP CHIP to identify gene copy number gain or loss. Alterations in the PI3K pathway were the most commonly identified molecular targets in HNSCC cell lines. Likewise, the PI3K signaling pathway is commonly altered in multiple cancers driving cancer cell growth and survival. We identified 5 cell lines with PIK3CA mutations, 5 cell lines with PIK3CA amplification, and 2 cell lines with PTEN loss. We tested these cell lines along with those lacking identified PI3K pathway alterations for sensitivity to two PI3K inhibitors (GDC0941, GSK1059615), one dual PI3K/mTOR inhibitor (GDC0980), one AKT inhibitor (GSK690693) and one mTOR inhibitor (AZD8055). The 5 cell lines with PIK3CA mutations were almost universally sensitive to all inhibitors whereas those with PI3KCA gene amplification were almost universally resistant to all inhibitors. Among two cell lines with PTEN loss, one was sensitive and other resistant to the inhibitors. Cell lines lacking identified PI3K pathway alterations had mixed sensitivity. We measured basal activation of several pathway components including pAKT, pS6, p4EBP1 and found that only pS6 predicted response to PI3K pathway inhibitors. Following treatment with PI3K inhibitors, we discovered an increase in pERK and insufficient inhibition of cMYC in resistant cell lines, but not in sensitive cell lines. The combination of PI3K and MEK inhibition was synergistic in resistant cell lines. Our study identified that among the different types genetic lesions in the PI3K pathway, PIK3CA mutations are the best predictors of sensitivity to pathway inhibitors. In resistant HNSCC combinations with MEK inhibitors may be effective. Multiple inhibitors of the PI3K and MEK/ERK pathways are in clinical development making the clinical application of our data possible in the near future. This work was supported by University of Texas SPORE in Head and Neck Cancer P50 CA097007. Citation Format: Tuhina Mazumdar, Lauren A. Byers, Shaohua Peng, Lixia Diao, Katherine S. Hale, Bonnie S. Glisson, Faye M. Johnson. Genetic determinants of sensitivity and resistance to PI3K inhibitors in head and neck cancers for efficient targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2388. doi:10.1158/1538-7445.AM2013-2388

Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC).In general, DNA repair pathway activation leads cellular resistant to DNA damaging agents. The loss of LKB1 leads NSCLC to be more invasive and resistant to DNA damage based therapies. We hypothesize that loss of LKB1 may contribute to DNA damage response. We investigated LKB1- mediated signaling pathways by stably overexpressing LKB1 in its mutated NSCLC cell lines, A549 and H460. Methods: Expression of 139 proteins was analyzed by reverse phase protein array (RPPA) in LKB1 overexpressing cell lines along with the appropriate vector control lines. Differences in protein expression at baseline in clones with LKB1 versus clones with vector control were assessed by unpaired T-test with Welch's correction. Ionizing radiation sensitivity (reproductive cell death) was assessed by clonogenic cell survival assay (CSA). Results: LKB1 overexpression in A549 and H460 showed a significant downregulation of genes involved in DNA damage and repair pathway, such as RAD50 (1.4-fold relative expression, p< 0.05 and 1.5-fold relative expression p Citation Format: Chao Yang, Uma Giri, Maria Angelica Cortez, Jayanthi Gudikote, YouHong Fan, Lixia Diao, Lauren A. Byers, John V. Heymach. LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2013-1589

The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2013-2498

Background: MEK inhibitors such as BAY86-9766 are a new class of agents that show promise in the treatment of non-small cell lung cancer (NSCLC). The downstream effects of MEK inhibition in NSCLC have not been fully elucidated. We performed a broad proteomic analysis to determine which signaling pathways were modulated by BAY86-9766 treatment and how these pathways correlate with sensitivity. Methods: We treated 109 lung cancer cell lines with MEK inhibitor BAY86-9766 at a concentration of 2200 nM. 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 1/3rd of those tested. 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: MEK inhibitor BAY86-9766 was effective in reducing phosphorylation of direct downstream signaling molecules pMAPK (p Conclusions: We have performed broad proteomic analysis on cell lines treated with MEK inhibitor BAY86-9766 to determine which pathways are modulated by treatment and how they might relate to sensitivity. We conclude that MEK inhibition with BAY86-9766 may exert some of its effects by suppressing mTOR activity via the LKB1/AMPK pathway, and that the degree of modulation of the AMPK pathway correlates with sensitivity. This mechanism may involve decreased activation of p90RSK by MAPK, which leads to decreased degradation and increased activity of LKB1, and thereby increased AMPK activity and decreased mTOR signaling. This works suggests a rational basis for combinations of targeted agents to overcome resistance, such as combinations of MEK inhibitors with mTOR inhibitors or PI3 kinase inhibitors. Citation Format: Kathryn A. Gold, Lauren A. Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai T. Tran, Kevin R. Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1045. doi:10.1158/1538-7445.AM2013-1045

10612 Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC) and functions as a tumor suppressor by activating AMPK. Loss of LKB1 by point mutation or deletion suppresses AMPK, leading to increased mTOR signaling. We investigated the signaling pathways modulated by LKB1 mutations and by mTOR inhibition in NSCLC. Methods: Protein expression in cell lines was measured by reverse phase protein array. Differences in protein expression at baseline in LKB1 wild-type versus mutant cell lines and the effects of protein modulation by treatment were assessed by ANOVA. Results: LKB1 mutant cell lines had lower expression of phosphorylated AMPK and TSC (p

7096 Background: Small cell lung cancer (SCLC) is an aggressive malignancy that differs from non-small cell lung cancer (NSCLC) in its metastatic potential and response to treatment. To date, no molecularly-targeted agent has prolonged survival of SCLC patients. Using a proteomic approach, we previously identified high expression of the DNA repair protein poly (ADP-ribose) polymerase 1 (PARP1) in SCLC cell lines and tumors. Here we test in vitro sensitivity of SCLC to PARP inhibition or knockdown. Methods: Cell lines were treated with PARP inhibitor olaparib or AG014699 for 14d +/- chemotherapy. Relative cell viability was assessed by cell count. siRNA against PARP1 was compared with scrambled siRNA and mock transfected cells. To assess DNA repair, RAD51 foci were counted after 1µM or 5µM olaparib and after 8Gy irradiation (RT). Results: SCLC cell lines were highly sensitive to PARP inhibition by olaparib (IC50s 8µM). Because BRCA1/2 and PTEN mutations are associated with greater sensitivity to PARP inhibitors, we compared SCLC sensitivity with that of BRCA1-mutated (HCC1395) and PTEN-mutated (MDA-MB-468) breast lines. As expected, HCC1395 and MDA-MB-468 were sensitive to both PARP inhibitors. Remarkably, however, SCLC cell lines were as sensitive or more so. Combination of olaparib with topotecan or irinotecan (commonly used in SCLC) decreased tumor cell viability more than either agent alone (p 4-fold) and RT (>18-fold). Conclusions: SCLC lines were as sensitive to PARP inhibition as BRCA1- or PTEN-mutated breast cancer lines. Moreover, PARP inhibition enhanced the effect of chemotherapy on SCLC lines. Increased formation of RAD51 foci in SCLC cells after olaparib or RT suggests a deficiency in homologous recombination that may account for the sensitivity to PARP inhibitors. These results support the investigation of PARP inhibition as a novel therapeutic approach in SCLC lung cancer.

Paclitaxel is a standard chemotherapeutic agent for ovarian cancer. Resistance of ovarian cancer cells to the drug has been a major obstacle in clinical practice. Thus, alternative approaches are needed to conquer the resistance. PEA-15 (phosphoprotein enriched in astrocytes-15 kDa) regulates cell proliferation and apoptosis. It is phosphorylated at S104 and S116 by Akt, PKC and CaMKII. PEA-15's functions are phosphorylation dependent. Although PEA-15 is known to mediate chemoresistance in breast cancer, the effect of PEA-15 phosphorylation status on chemosensitivity remains unknown. We hypothesized that phospho-PEA-15 (pPEA-15) enhances sensitivity of ovarian cancer cells to paclitaxel. To test our hypothesis, we silenced PEA-15 expression in HEY and OVTOKO cells using siRNA and observed a 14% reduction in apoptosis after paclitaxel exposure. To further determine if PEA-15 phosphorylation contributes to chemosensitivity, we generated SKOV3.ip1-vector (control), SKOV3.ip1-AA (AA, phosphoinhibitory at S104 and S116) and SKOV3.ip1-DD (DD, phosphomimetic at S104 and S116) stable ovarian cancer cells. Compared to the control, DD cells showed a 15% reduction in cell viability (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5670. doi:1538-7445.AM2012-5670

Background: MPM is caused by lethal neoplastic growth of the pleura surrounding lungs. It is resistance to most standard anti-cancer treatment regimens and needs discovery of newer therapeutic approaches. MPM is characterized by massive loco-regional invasion of the malignant pleural cells into the lung parenchyma. Twist1 is a transcription factor, which promotes invasion and metastasis of tumor cells, increases chemotherapeutic resistance and is involved in the pathobiology of many cancers. Also recent studies have highlighted the potential of twist1 as a therapeutic target in cancer. But there is no report investigating its function in mesothelioma. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Bioinformatic analysis of the microarray data using a 2 sample t-test was applied, on a probe-by-probe basis followed by Beta-uniform Mixture for multiple comparisons, to determine the differences between tumor vs normal specimens. The microarray results were validated by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) using Taqman assays on the ABI 7300 platform. For all qRT-PCR experiments the twist1 transcript levels were determined relative to endogenous GAPDH as control using ΔΔCT calculation. We performed Western blot analysis on a panel of 16 mesothelioma cell lines including Met-5A (SV-40 immortalized) and HCT-4012 (telomerase immortalized) pleural mesothelial control cell lines. Results: The bioinformatic analysis of microarray data showed that twist1 transcript level was 8.7 fold higher in tumors (p = 1.1E-16) compared to paired normal specimens. Using qRT-PCR, we compared twist1 transcript levels in 12 pairs of tumor vs paired normal tissue specimens and found that twist1 was upregulated to more than 10 fold in MPM tumors (p < E-4). Western blot showed that 10 MPM cancer cell lines had higher expression of twist1 protein compared to Met-5A and HCT-4012 cell lines. The highest expression was seen in 2 of the sarcomatoid cell lines - RS5 and DM3, suggesting a correlation with metastatic phenotype since sarcomatoid tumors are highly metastatic in nature. Conclusion: Our preliminary findings suggest that twist1 is upregulated in MPM tumors and cell lines and may play a role in the development of MPM. Further studies are needed to investigate its role in the process of tumorigenesis and metastasis. Supported by Grants: DoD W81XWH-07-1-0306 (I.I.W and AST), Fleming Foundation, IASLC Young Investigator Award 2011-2013 (MS). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3404. doi:1538-7445.AM2012-3404

Background: LKB1 and KRAS are the most commonly mutated genes in non-small cell lung cancer (NSCLC), each present in up to 35% of patient tumors. KRAS mutations have been associated with resistance to EGFR tyrosine kinase inhibitors, but it is not established to what extent KRAS or LKB1 mutations may predict response to other systemic treatments, including PI3K/Akt and MEK inhibitors. Methods: IC50s for cytotoxic chemotherapies and targeted drugs were determined by MTS assay in a large panel of NSCLC cell lines with known mutational status. Cells lines with and without mutations were compared by t-test and linear mixed model to determine the effect of single and double mutations. Protein expression in cell lines was measured by reverse phase protein array. Results: LKB1 mutations strongly predicted resistance to Akt inhibition by MK2206 (p=0.004), but not to PI3K inhibitors BAY80-6949, GDC0941, or 8-amino-adenosine (p>0.24). In contrast, KRAS mutations were strongly associated with resistance to PI3K inhibitors BAY80-6949 (p≤0.0001) and GDC0941 (p=0.034) and to Akt inhibition by MK2206 (p=0.047). Conversely, there was a trend towards greater sensitivity in the KRAS mutated cells to the MEK inhibitor BAY86-9766. Resistance to PI3K inhibition in KRAS mutated lines was largely abrogated by the combination of BAY80-6949 and BAY86-9766. Co-existing LKB1 and KRAS mutations were present in 18% of cell lines, but were not significantly more resistant to PI3K/Akt inhibitors, nor did they predict greater MEK inhibitor sensitivity. There was no association between KRAS or LKB1 mutations and response to cytotoxic chemotherapies, including docetaxel, pemetrexed, or platinum doublets. At the protein level, LKB1 mutant cell lines had significantly higher expression of IGF1R (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5605. doi:1538-7445.AM2012-5605

Lung cancer is the leading cause of cancer related mortality in the US of which more than 75% cases are that of non-small cell lung carcinoma (NSCLC). Mutations in the proto-oncogene KRAS have been linked with poor prognosis for NSCLC patients. In this study, we aimed at analyzing the relationship between specific KRAS mutations and NSCLC cell radiosensitivity and protein expression patterns. We analyzed 22 NSCLC cell lines and stratified them according to their KRAS status. Our results show that NSCLC cells harbouring G12C mutations are more sensitive to radiation compared to cells with other KRAS status (SF2 = 0.35 ± 0.16 for G12C vs. 0.63 ± 0.17 for other KRAS mutants vs 0.54 ± 0.15 for wt KRAS). Our protein expression data suggests that G12C mutants have reduced expression of DNA-repair proteins such as ATM, Rad 50, Ku 80 and XRCC1 (p < 0.05) which may be responsible for their radiosensitive nature. Further we found that G12C mutant NSCLC cell lines have reduced expression of pAkt compared to cells with other KRAS status (p =0.009). This suggests that NSCLC cells harboring G12C mutation would be less susceptible to PI3K inhibition induced radio-sensitization. Our clonogenic assay results confirm our finding when 1-hour pretreatment with 5 µM LY294002 (PI3K inhibitor) sensitized H460 and A549 (non-G12C mutated NSCLC cells) but not H1792 and H23 (G12C mutant NSCLC). Our results strongly suggests that G12C KRAS mutant cells are radio-sensitive compared to NSCLC cells of other KRAS status and PI3K inhibitor therapy along with radiation could be beneficial for patients harboring non-G12C mutational KRAS status. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3454. doi:1538-7445.AM2012-3454

Background: MPM is a lethal neoplasm of the pleural layer of cells surrounding lungs and is in dire need of newer therapeutic treatments. microRNA's (miRs) play a critical role in the pathobiology of many cancers but there are few reports investigating their function in MPM. Recently many studies have highlighted the potential of miRs as therapeutic agents in cancer. We completed a microarray profiling strategy to discover miRs of therapeutic and biological importance in MPM. Methods: We extracted total RNA from 53 frozen resected tumor tissue specimens, comprised of 39 epitheloid, 7 sarcomatoid and 7 biphasic histotypes, along with paired normal tissue. The RNA was labeled and hybridized to Agilent v3 Human miR microarrays. These were scanned and the data was processed using the AgiMicroRna “R” package involving background correction, quantile normalization and summarization. The microarray results were validated by quantitative reverse transcription polymerase chain reaction (qRTPCR) using Taqman assays on the ABI 7300 platform. Also qRT-PCR was employed to determine the levels of miR-203 expression in a panel of 26 mesothelioma cell lines including Met-5A, an SV-40 immortalized pleural mesothelial control cell line. For all qRT-PCR experiments the miR-203 levels were determined relative to endogenous miR-U6 as control using ΔΔCT calculation. Moreover in a previous study RNA from these same tissue specimens were hybridized on Affymetrix U133 plus 2.0 microarray to obtain transcriptomic profiles. Results: The bioinformatic analysis of miR microarray data showed that a number of miR's, including miR-203, are differentially expressed between the tumor and normal samples. A paired T-test conducted on a miRNA-by-miRNA basis and at a highly significant FDR value of 1e-06 showed miR-203 to be down regulated, more than 2 fold, in tumors compared to paired normal tissue. We decided to explore the role of miR-203 in the pathogenesis of MPM since it has been postulated to play a tumor suppressor role in skin and prostate cancer by inhibiting proliferation, metastasis and acting antagonistic to stem cells (1, 2). Using qRT-PCR, we compared levels of expression in 40 pairs of tumor vs. paired normal tissues and demonstrated that miR-203 was down regulated to more than 10 fold in MPM tumors. Also qRT-PCR showed that 70 % of 25 MPM cancer cell lines had lower expression of miR-203 compared to Met-5A cell line. Our previous transcriptomic profiling study had shown differentially expressed transcripts between these same tumors and paired normal specimens. Survivin (BIRC5) message level, which codes for an apoptosis inhibitor protein and reported to be regulated by miR-203 in prostate cancer cell lines (3), was found to be 2.7 fold higher in tumors (p = 2.00e-15) in an expected anti-correlation direction to miR-203 expression levels (i.e. low miR = high target mRNA). Conclusion: Our findings suggest that miR-203 may play a tumor suppressor role in pleural mesothelioma and regulate levels of survivin message. Supported by Grants: DoD W81XWH-07-1-0306 (IW and AT), Fleming Foundation, IASLC Young Investigator Award 2011–2013 (MS).

Abstract 238 Survivin, a member of the inhibitor of apoptosis (IAP) protein family, plays important roles in cell proliferation and survival and is highly expressed in various malignant cells, including leukemic cells. We showed previously that targeting survivin expression in AML cells induces a cell proliferation defect and subsequent cell death involving the mitochondrial pathway. To assess its usefulness as a prognostic marker in AML, we profiled survivin expression in samples from 511 newly diagnosed AML patients using a validated reverse-phase protein array and correlated its protein expression levels with clinical outcomes. We found that survivin levels were higher in the bone marrow (BM) than in peripheral blood (PB) leukemic cells in 140-paired samples (p = 0.0001) consistent with its function in cell proliferation. Survivin levels did not correlate with cytogenetic groups, the mutational status of nucleophosmin or FLT-3, BM or PB blast counts, white blood count, or patient performance status. Interestingly, survivin levels were found to significantly predict shorter overall (P = 0.016) and event-free (P = 0.023) survival in multivariate Cox model analysis. Age, gender, white blast count, BM blasts, cytogenetics, and survivin levels were included in the final model. The groups expressing low and high survivin were defined by the median value. This was also true when data sets from BM and PB were analyzed separately. Further, we found that survivin levels were significantly higher in CD34+38− AML stem/progenitor cells than in bulk AML blasts and total CD34+ cells (2.06 and 1.91 fold, respectively, P < 0.001) when we analyzed survivin expression in 37 samples of isolated CD34+38− AML cells (see Figure). These results suggest that survivin expression levels are prognostic in AML and that survivin is overexpressed in AML stem/progenitor cells, may play an important role in AML stem cells, and may thus be an important target in AML therapy. Disclosures: No relevant conflicts of interest to declare.

Background: Epithelial/mesenchymal transition (EMT) is associated with loss of cell adhesion molecules such as E-cadherin and increased invasion, migration, and proliferation in epithelial cancers. In non-small cell lung cancer (NSCLC), EMT is associated with greater resistance to EGFR inhibitors. However, its potential to predict response to other targeted drugs or chemotherapy has not been well characterized. Using a previously-derived EMT gene signature, we tested the association between EMT and drug response in a panel of >50 NSCLC cell lines. Materials/Methods: NSCLC cell lines were classified as epithelial or mesenchymal by a previously described 76-gene EMT gene expression signature. IC50s for more than 30 drugs or drug combinations were determined by MTS assay. Differences in IC50 between epithelial and mesenchymal lines were assessed by t-test (p-values ≤ 0.05 were considered significant). Results: As expected, EGFR mutated cell lines were classified by the EMT signature as epithelial and were highly sensitive to EGFR inhibition by erlotinib. Among wild-type EGFR cell lines, those that had undergone EMT were significantly more resistance to erlotinib (p=0.029). NSCLC cell lines with mesenchymal signatures were also more resistant to drugs targeting the PI3K/Akt pathway such as GDC0941 (p=0.037) and 8-amino-adenosine (p=0.009). A trend towards greater resistance was also seen in mesenchymal cells treated with the Akt inhibitor MK2206 (p=0.12). There was no association between EMT and response to cytotoxic chemotherapies, including cisplatin, carboplatin, gemcitabine, pemetrexed, docetaxel, paclitaxel, and platinum-doublets (p-values ≥0.2). Conclusion: EMT is associated with greater resistance to PI3K/Akt pathway inhibitors and may be a useful predictive marker for patients treated with these drugs. The EMT signature is being investigated prospectively in patients treated with an Akt inhibitor in an ongoing clinical trial. Supported in part by the NIH Lung Cancer SPORE grant P50 CA70907. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B156.

7010 Background: Improved predictions for survival and response to therapy will help to refine clinical care and potentially improve outcomes for patients with cancers of the upper aerodigestive tr...

Background: Despite a low response rate, erlotinib (E) improves survival in a subset of NSCLC pts with wt EGFR but there are no established markers for identifying pts likely to have clinical benefit. We hypothesized that a gene expression sig could be used for this purpose. Material and Methods: We used pretreatment gene expression profiles (Affymetrix HG1.0ST) from 101 chemo-refractory pts in our Biomarkers-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) treated with E, E+bexarotene (EB), sorafenib (S), or vandetanib (V). 24 cases of wt EGFR & KRAS tumors treated with E or EB were compared to train the signature (two-sided t-test), using the primary end-point of the trial [8-week disease control (8wDC)]. Principal component (PC) analysis and a logistic regression model were used to develop the sig. Gene expression profiles from 108 NSCLC cell lines (Illumina), with available E IC50 (N=94) and DNA methylation profiling (N=66, Illumina), were used for in vitro studies. Results: 113 genes were differentially expressed between pts with or without 8wDC (false discovery rate 30%; P=0.004). Leave-one-out cross validation with various gene list lengths produced a 5-gene sig, including lipocalin 2 (LCN2), with a specificity, sensitivity and accuracy of 80% to predict 8wDC. In pts treated with E or EB, using the median sig score, the 8wDC rate in the sig-positive group was 83% compared with 0% in the sig-negative group; the sig did not predict 8wDC in pts treated with S or V (Mantel-Haenszel chi-squared test P=0.023). The improvement in 8wDC in the sig-positive group translated to an increased progression-free survival (PFS) (hazard ratio=0.12, 95% confidence interval: 0.03-0.46, P=0.001; log-rank P=0.0004; median PFS: 12.5 weeks vs. 7.2 weeks). We tested the sig in an independent set of 47 wt EGFR&KRAS cell lines. It predicted E sensitivity with an area under the curve of 78% (P=0.002). The first PC of the sig and the IC50 for E were correlated (r=−0.47, P=0.0009). In 108 NSCLC cell lines, LCN2 gene expression was bimodal and correlated with the IC50 for E (r=−0.46, P=0.001). Degree of methylation and expression level of LCN2 were inversely in wt EGFR & KRAS NSCLC cells (r=−0.79, P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4109. doi:10.1158/1538-7445.AM2011-4109

Background: There are currently no established markers to identify pts bearing wild-type EGFR who are likely to benefit from erlotinib (ERLO). The EGFR and Kras pathways, and epithelial to mesenchymal transition (EMT), have been associated with response/resistance to EGFR inhibitors. We developed gene signatures for these pathways and tested whether they were predictive of disease control (DC) and tumor mutations using gene expression profiles from pts in the BATTLE trial, and developed novel markers for ERLO benefit in wt EGFR pts. Methods: Gene expression profiles (Affymetrix HG1.0ST) from pretreatment core needle biopsies (CNBs) were obtained from 101 BATTLE pts. Pathways signatures were developed using independent datasets from resected NSCLC pts and cell lines. A robust EGFR mutation signature was derived by comparing genes differentially expressed in mutated and wt EGFR lung adenocarcinoma from 3 independent institutions, and validated in three independent sets, both in vivo and in vitro. A KRAS signature was similarly derived. An EMT signature was derived by identifying genes with a bimodal distribution and correlated with known EMT genes (E-cadherin, vimentin, N-cadherin, FN-1) using 54 NSCLC cell lines, and validated in an independent panel of HN cell lines and across different platforms. A novel 5-gene signature was derived using erlotinib-treated BATTLE patients with or without 8 week DC, the primary study endpoint. Results: The EGFR and Kras signatures predicted EGFR and Kras mutations, respectively, in BATTLE patients (AUC 0.72 by ROC analysis, p=0.03 for EGFR; AUC 0.67, p=0.0.01 for KRas signature). In pts with wt EGFR and Kras, the EMT and 5-gene, but not the EGFR or KRas signatures, were associated with improved DC in ERLO treated pts (EMT signature: 64% for epithelial vs 10% mesenchymal groups, p=0.02; 5-gene: 83% vs 0%, p= Conclusions: Gene expression profiling from CNBs is a feasible approach for predicting response and identifying activated oncogenic pathways and potential therapeutic targets in refractory NSCLC pts. EGFR and Kras signatures predicted mutation status but, in wt EGFR patients, did not predict efficacy. EMT and a novel 5-gene signature including LCN2/NGAL were predictive of DC in pts with wt EGFR treated in BATTLE and merit further investigation as markers of benefit for EGFR inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-88. doi:10.1158/1538-7445.AM2011-LB-88

This is an open access article. Unrestricted non-commercial use is permitted provided the original work is properly cited. The reproducibility of mass spectrometry (MS) data collected using surface enhanced laser desorption/ionization-time of flight (SELDI-TOF) has been questioned. This investigation was designed to test the reproducibility of SELDI data collected over time by multiple users and instruments. Five laboratories prepared arrays once every week for six weeks. Spectra were collected on separate instruments in the individual laboratories. Additionally, all of the arrays produced each week were rescanned on a single instrument in one laboratory. Lab-to-lab and array-to-array variability in alignment parameters were larger than the variability attributable to running samples during different weeks. The coefficient of variance (CV) in spectrum intensity ranged from 25% at baseline, to 80% in the matrix noise region, to about 50% during the exponential drop from the maximum matrix noise. Before normalization, the median CV of the peak heights was 72% and reduced to about 20% after normalization. Additionally, for the spectra from a common instrument, the CV ranged from 5% at baseline, to 50% in the matrix noise region, to 20% during the drop from the maximum matrix noise. Normalization reduced the variability in peak heights to about 18%. With proper processing methods, SELDI instruments produce spectra containing large numbers of reproducibly located peaks, with consistent heights.

MPM is a lethal neoplasm exhibiting low median survival of patients and lacks effective therapeutic options. There is an urgent need to understand the underlying pathobiology and discover novel therapeutic targets. We undertook a messenger RNA (mRNA) expression profiling strategy to determine the pathways and biomarkers significantly altered in MPM tumors. We isolated total RNA from 55 MPM tumors with 38 paired controls representing 39 epitheloid, 8 biphasic and 6 sarcomatoid cases. The paired controls were adjacent non-tumor tissue samples and histopathological analysis revealed a tumor content of greater than 70 % in most of these tumors cases. The RNA was labeled and hybridized onto Affymetrix U133 Plus 2.0 chips to ascertain the global expression profile in these tumors. Bioinformatic analysis of the microarray data using a two sample t-test was applied on a probe-by-probe basis followed by Beta-uniform Mixture for multiple comparisons. Finally paired t-test was applied to determine the differences between tumor vs normal samples. About ∼955 highly significant probesets representing ∼ 670 genes, at a FDR of e-09, were obtained and subjected to pathway analysis using MetaCore software suite (GeneGo, Inc.). The most significantly altered pathway in MPM tumors was the Mitotic Spindle Assembly Checkpoint (MSAC) pathway due to up-regulation of at least 15 genes including a ∼3.4 fold increase in Aurora kinase A, which is currently being explored in other cancers as a potential therapeutic target. The other genes belonging to this pathway, also up-regulated in tumors, include Mad2L1 and BUBR1 which together regulate cell division cycle 20 (Cdc20) protein, an essential cofactor needed by the Anaphase Promoting Complex to initiate the anaphase of cell cycle. Interestingly we also discovered that some of the MSAC pathway genes show a histotype-specific graded expression pattern with higher levels in sarcomatoid tumors compared to biphasic tumors and with lowest expression levels seen in epitheloid tumors. Additionally survivin, the product of which localizes to the mitotic spindle and negatively regulates apoptosis by inhibiting caspase activation, was expressed more than 2 fold in MPM tumors compared to normal samples. The microarray data also revealed other pathways significantly upregulated in MPM tumors including the Wnt and Cell-Adhesion signaling pathways. We are currently validating the expression microarray data using quantitative Polymerase Chain Reaction (PCR) platform with respect to key components of MSAC pathway. This will be followed by proteomic analysis of these components on tumor lysates to confirm the alterations in their expression profiles. Supported by Grants: DOD PROSPECT W81XWH-07-1-030602, Fleming Foundation for Mesothelioma Research, Aileen Dillon and Lee Bourg Mesothelioma Endowment and NIH K12 CA088084 08 award. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2172.

Additional file 1. Supplementary Data: Table S1. Spearman’s correlation between OX-40 and other IHC immune markers. Figure S1. Overall survival Kaplan-Meier curves of OX-40 protein expression in squamous cell carcinoma histology (A) and adenocarcinoma histology (B) by median value. Figure S2. Overall survival Kaplan-Meier curves by OX-40 IHC level and CD3/CD8/OX-40 level (A), ICOS/OX-40 level (B), PD-L1/OX-40 level (C). (PDF 198 kb)

Additional file 1. Supplementary Data: Table S1. Spearman’s correlation between OX-40 and other IHC immune markers. Figure S1. Overall survival Kaplan-Meier curves of OX-40 protein expression in squamous cell carcinoma histology (A) and adenocarcinoma histology (B) by median value. Figure S2. Overall survival Kaplan-Meier curves by OX-40 IHC level and CD3/CD8/OX-40 level (A), ICOS/OX-40 level (B), PD-L1/OX-40 level (C). (PDF 198 kb)