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Background: We have previously demonstrated that the presence of a proinflammatory peripheral cytokine milieu correlates with high levels of circulating neutrophils at the time of surgery and reduced overall survival and recurrence-free survival. In this study, we hypothesized that functional immune features or states in circulation may indicate early tumor recurrence when assessed longitudinally. We also investigated whether potential deficiencies in peripheral immune functionality at the time of lung cancer resection could identify correlates with subsequent outcome. Methods: We performed flow cytometry and luminex profiling of blood samples collected from patients with stage I-IIIA resected NSCLC (n=150) and enrolled on the Immunogenomic profiling of NSCLC (ICON) prospective protocol. Patient characteristics include 75 adenocarcinomas, 30 squamous and 12 mixed or other histologies. Only patients who underwent primary cancer resection without neoadjuvant therapy were included. At a median follow up of 18.2 months, 37 patients had disease recurrence. Blood was collected at the time of primary lung cancer resection, at 4 weeks, and 4 months thereafter with PBMCs utilized for flow cytometry and plasma for cytokine assessment. Changes in cytokines were assessed by normalizing to baseline levels. Results: Larger tumors as well as advanced clinical and pathological stages were associated with higher frequencies of proliferating Ki67+CD4+ and Ki67+CD8+ T cells in circulation at time of resection, suggesting an activated circulating immune response. We identified novel strong correlations in the plasma between soluble BTLA and Tim3 (r= 0.87, p=1.74e-140), PD1 and CD80 (r=0.72, p=6.41e-74) and moderate correlations between soluble PD1 and PDL1 (r=0.39, p=8.41e-18). Efforts are ongoing to determine the association of specific circulating immune states with the presence of these soluble receptors. CD8+Tim3+ T cells (FC=5.2%, p=0.028) and CTLA4+NK cells (FC=47.7%, p=0.002) as well as CTLA4+Tregs (FC=25.2%, p=0.05) were found to be increased in circulation at pre-recurrence time points (either 4 weeks or 4 months) relative to resection. This suggests the emergence of a suppressive cell type as well as induction of specific checkpoint receptors on effector cells that correlate with tumor recurrence. Finally, we identified a cytokine signature associated with recurrence by testing three sets within the ICON cohort with a training set AUC = 0.76 and the test set AUC=0.72, which was validated in a third set of patients yielding an AUC = 0.76. Conclusions: We identified circulating immune features associated with initial tumor size and overall stage as well as unique associations among soluble proteins. Increased presence of potentially inhibited or suppressed CD8+ T cells and NK cells as well as Tregs are suggestive of mechanisms of immune suppression relative to tumor recurrence. Citation Format: Younghee Lee, Daniel McGrail, Hai Tran, Mayra E. Vasquez, Carlos Ramos, Alexandre Reuben, Ara A. Vaporciyan, Annikka Weissferdt, Chantale Bernatchez, Tina Cascone, Ignacio I. Wistuba, Jianjun Zhang, John Heymach, Marcelo V. Negrao, Don L. Gibbons, Boris Sepesi, Cara L. Haymaker. Circulating biomarkers are associated with recurrence following complete resection of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1670.

Background: Tumor genomic information from tissue has been the standard of practice for identifying actionable molecular alterations. The same genomic profiling is also widely available by a non-invasive blood test from circulating tumor DNA (ctDNA). We hypothesized that patients with advanced non-small cell lung cancer (NSCLC) with actionable oncogenic driver mutations with either low or high variant allelic frequency (%VAF) identified by ctDNA would have similar clinical benefits after treatment with targeted therapies. Methods: In this retrospective study (UTMDACC IRB-approved protocol PA16-0061), patients with a targetable mutation identified by ctDNA NGS assay who received an FDA-approved therapy were identified in our GEMINI database. All potential patients were individually reviewed and information confirmed with review of their electronic medical records. Blood based genomic profiling was completed by NGS of ctDNA with a panel of up to 70 cancer-related genes at a CLIA-certified lab (Guardant360, Guardant Health, Redwood City, CA). The %VAF was reported for each mutation identified. We defined low group with VAF of 0.01% to 0.99% and high group with VAF > 1%. Tumor measurements were assessed using RECIST V1.1. For comparisons analysis, Kaplan-Meier estimator was used to estimate progression free survival (PFS) with Mantel-Cox and un-paired t-test was used for tumor responses. Results: Eighty-nine patients with advanced NSCLC treated with an FDA approved therapy based on ctDNA genomic profiling were identified and the results are summarized in table below: LOW %VAFHIGH %VAFP valuegroup (n=39)group (n=50)Gender (Female/Male)20/1928/22Age (median, range)54(27-85)63(29-89)Targetable mutations:EGFR2246ALK114MET exon 1420ROS120BRAF20Line of therapy (median, range)2(1-7)2(1-7)ALL PatientsTumor response (mean, range)-37.23% (-100 to +38)-31.08% (-100 to +59)0.42PFS (median, range)215 (27 – 848) days280 (43 – 894) days0.70EGFR onlyTumor response (mean, range)-24.86% (-100 to +16)-23.54% (-100 to +59)0.88PFS (median, range)189 (57 – 848) days293 (43-894) days0.23 Conclusions: In this analysis of patients who were treated with an FDA approved therapy based on genomic results from ctDNA profiling, there were no differences in either tumor response (P value = 0.42) or PFS (P value = 0.70) between those patients with low and high ctDNA as assessed by %VAF of identified targetable mutation. Similar findings were also observed in the analysis of the EGFR only subgroup. This study reveals patients with low %VAF received comparable clinical benefits as those with high %VAF and no deleterious harm was observed from treating patients with low %VAF with appropriate targeted therapy. Citation Format: Hai T. Tran, Vincent K. Lam, Mayra Vasquez, Yasir Elamin, Victoria Raymond, Lingzhi Hong, George Blumenschein, Brett Carter, Richard Lanman, Mehmet Altan, Ann Tsao, Lauren Byers, Don Gibbons, Jianjun Zhang, John V. Heymach. Comparable clinical benefits between low and high variant allelic frequency in NSCLC patients treated with targeted therapies based on molecular alterations from ctDNA NGS [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 787.

Non–small cell lung cancer (NSCLC) cells with somatic mutations in K-ras recruit to the tumor a variety of cell types (hereafter collectively termed “stromal cells”) that can promote or inhibit tumorigenesis by mechanisms that have not been fully elucidated. Here, we postulated that stromal cells in the tumor microenvironment alter the tumor cell secretome, including those proteins required for tumor growth and dissemination, and we developed an in vitro model to test this hypothesis. Coculturing a murine K-ras mutant lung adenocarcinoma cell line (LKR-13) with a murine lung stromal cell (macrophage, endothelial cell, or fibroblast) enhanced stromal cell migration, induced endothelial tube formation, increased LKR-13 cell proliferation, and regulated the secretion of proteins involved in angiogenesis, inflammation, cell proliferation, and epithelial-to-mesenchymal transition. Among these proteins, CXCL1 has been reported to promote NSCLC development, whereas interleukin-18 (IL-18) has an undefined role. Genetic and pharmacologic strategies to inhibit CXCL1 and IL-18 revealed that stromal cell migration, LKR-13 cell proliferation, and LKR-13 cell tumorigenicity required one or both of these proteins. We conclude that stromal cells enhanced LKR-13 cell tumorigenicity partly through their effects on the secretome of LKR-13 cells. Strategies to inhibit tumor/stromal cell interactions may be useful as therapeutic approaches in NSCLC patients. [Cancer Res 2008;68(17):7237–45]

PURPOSE: Host immune activation is believed to be a major player of radiation pneumonitis (RP). Several cytokine and angiogenic factors (CAFs) have been studied as correlative markers and therapeutic targets for RP; however, studies are sparse in the setting of prospective trials and high-grade toxicity (Grade≥3) endpoints. We examined the predictive value of several CAFs in combination with clinical factors for RP in the context of a Phase II, prospective randomized study of intensity modulated RT (IMRT) versus proton therapy for locally advanced NSCLC (n=178) as well as a prospective laboratory cohort (n=43). METHODS: From July 2009 to April 2014, 221 patients were treated with proton therapy or IMRT to doses of 60-74 Gy equivalents with concurrent platinum-doublet chemotherapy. Primary endpoints were symptomatic RP (Grade≥2) and RP requiring intervention (Grade≥3). Plasma circulating levels of IL-6, IL-10, IL-8, IL-17, IL-2, IL-1β, and IL-1α were measured at baseline and post-treatment (median time 48 days post-RT) by multiplex electrochemiluminescence immunoassay. Clinical characteristics were evaluated for associations with Grade≥3 RP: histology, age, smoking status, mean lung dose (MLD), chemotherapy, stage, performance status, and RT technique. CAFs with >50% undetectable samples were removed from analysis, and samples below the lower limit of quantitation (LLQ) were transformed to the mean of zero and the LLQ. Predictive values of clinical and cytokine factors were evaluated in univariate and multivariate analyses via Cox proportional hazards modeling. RESULTS: Of the 221 patients, 42 (19.0%) developed Grade 2 RP, and 22 (9.95%) developed Grade≥3 RP. Both IL-1α and IL-1β had >50% of undetectable samples and were removed from analysis. Pre-treatment level of IL-6 ≥4.00 pg/mL was associated with Grade≥3 RP (HR 2.487; p=.048), as was post-treatment level of IL-10 ≥0.50 pg/mL (HR 2.931; p=.020). On multivariate analysis, the factors associated with Grade≥3 RP development were post-treatment level of IL-10 ≥0.50 pg/mL (HR 3.332; p=.015) and MLD ≥20 Gy (HR 5.864; p CONCLUSIONS: In this preliminary analysis of prospectively collected CAFs, post-radiation IL-10 level was significantly associated with Grade≥3 RP and, if validated, may serve as a predictor of this toxicity in combination with MLD. IL-10 plays an important role in the inflammatory response and is potentially targetable, warranting further investigation. Citation Format: Matthew S. Ning, Ting Xu, Daniel Gomez, Hai T. Tran, Ritsuko Komaki, Xiong Wei, Steven H. Lin, Stephen Hahn, Eric Prado, Jianjun Zhang, John V. Heymach, Zhongxing Liao. Circulating cytokine levels predictive of Grade≥3 radiation pneumonitis: Preliminary secondary analysis of a randomized controlled trial of definitive chemoradiation in locally advanced NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4745. doi:10.1158/1538-7445.AM2017-4745

Introduction: We present serum correlative analysis from a phase II randomized trial to identify markers associated with progression free survival (PFS) in NSCLC. Methods: NSCLC patients presenting with oligometastatic disease (≤3 metastases) were enrolled on a multi-institution phase II randomized trial (NCT01725165). Patients who did not progress after ≥3 months of first-line systemic therapy were randomized to upfront local consolidative therapy (LCT arm) to all sites of disease or at later progression (no-LCT arm). For correlative analyses, peripheral blood was collected from consenting patients after completion of initial systemic therapy but prior to randomization (baseline) and at each follow up. Serum cytokine and angiogenesis factors (CAFs) were analyzed via magnetic bead-based multiplex assay (Merck Millipore). Peripheral blood T cell receptor (TCR) repertoire was profiled by immunoSEQ (Adaptive Biotechnologies). Peripheral cell-free DNA (cfDNA) was subjected to next generation sequencing of a panel of cancer genes. Comparisons of PFS, the primary endpoint of this study, were conducted via log-rank test. Results: After enrollment of 74 patients with a median follow up of 19 months, this study was closed early after the Data Safety Monitoring Committee review determined efficacy in the LCT arm. Of 49 randomized patients, correlative blood draws were conducted on 33 patients. Considering all patients, high baseline EGF (cutpoint 50 pg/ml; median PFS: 5.7 vs 19.8 mo), Eotaxin (cutpoint 110 pg/ml; median PFS: 5.7 vs 21 mo), TNF alpha (cutpoint 8 pg/ml; median PFS: 3.6 vs 17.9 mo), or low baseline GRO (cutpoint 600 pg/ml; median PFS: 17.9 vs 3.6 mo) were associated with longer PFS (all P Conclusions: In this exploratory analysis, we identified baseline CAFs associated with PFS. These finding are being validated in a larger cohort of patients. If validated, CAFs could be a biomarker to select patients with oligometastatic NSCLC for aggressive local treatment. Citation Format: Chad Tang, John V. Heymach, Alexandre Reuben, Hai Tran, James Welsh, Jennifer Wargo, Zhongxing Liao, Anne Tsao, Eric Prado, Stephen Swisher, Jianjun Zhang, Daniel Gomez. Correlative analysis of systemic markers from a randomized trial assessing local consolidative therapy for non-small cell lung cancer (NSCLC) patients with oligometastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2774. doi:10.1158/1538-7445.AM2017-2774

Therapeutic strategies to target EGFR tyrosine kinase inhibitor (TKI) resistance mediated by mechanisms other than T790M are a major clinical challenge. Studies have implicated IL-6 as a mediator of EGFR TKI resistance, and IL-6 is known to be regulated by adrenergic receptors (AR) in some cancers. We investigated whether adrenergic pathways can promote T790M-independent EGFR TKI resistance in preclinical models and in clinical studies. We found that β2-AR was highly expressed in our panel of 119 cell lines and in NSCLC clinical specimens. Activation of β-ARs by stress hormones such as norepinephrine (NE) induced a dramatic rise in IL-6, and this occurred through β2-ARs. β-AR inhibitors (i.e. propranolol), but not α-AR inhibitors, blocked IL-6 induction. Analysis of downstream signaling pathways revealed that β2-ARs induced IL-6 expression through activation of adenylyl cyclase, p90RSK and CREB. To identify novel signaling pathways modulated by ARs, we stimulated NSCLC cell lines with NE and analyzed protein lysates by RPPA to detect expression and activation of >100 proteins. β-AR signaling inactivated the tumor suppressor LKB1 through phosphorylation of S428 and subsequently increased mTOR activity. LKB1 inactivation was critical for IL-6 induction. This finding is important as LKB1 loss is known to be a driver of NSCLC resistance and metastasis. Moreover, we found that β2-AR activation promoted EGFR TKI resistance in cell lines and in mouse models of EGFR mutant NSCLC. The effect of β-AR on EGFR TKI resistance was blocked by the addition of the beta blocker propranolol or IL-6 antibodies, in vitro and in vivo. Consistent with our preclinical studies, in the phase III ZEST clinical study testing erlotinib vs vandetanib, we found that high plasma levels of IL-6 was associated with a worse PFS and OS in the erlotinib arm. In addition, we found that circulating levels of IL-6 were significantly lower in NSCLC patients incidentally receiving beta blockers in the BATTLE trial. Finally, we analyzed the influence of incidental beta blocker use in the LUX-Lung3 study testing afatinib vs chemotherapy in EGFR mutant NSCLC patients. In patients not receiving beta blockers, the median PFS was 11.1 and 6.9 months for afatinib and chemotherapy, respectively, and afatinib improved PFS with a hazard ratio (HR) of 0.60. In patients receiving beta blockers, the median PFS was 13.6 and 2.5 for afatinib and chemotherapy, respectively, and afatinib improved PFS with a HR of 0.25. In conclusion, our preclinical and clinical data provide evidence that β2-AR activation can upregulate IL-6 in EGFR mutant+ NSCLC, modulate the LKB1/AMPK/mTOR axis, and promote EGFR TKI resistance. Moreover, EGFR mutant+ patients using beta blockers had greater relative PFS benefit from afatinib vs chemotherapy compared with those not using beta blockers, supporting future clinical testing of EGFR TKIs in combination with beta blockers. Citation Format: Monique B. Nilsson, Huiying Sun, Lixia Diao, Pan Tong, Youhong Fan, Hai Tran, Diane Liu, Guillermo Armaiz Pena, Jing Wang, Phil Rowe, Alan Webster, Jack Lee, Daniel Gomez, Waun Ki Hong, Ignacio Wistuba, Anil Sood, John Heymach. Beta blockers abrogate EGFR TKI resistance induced by adrenergic receptor-mediated upregulation of IL-6 and modulation of the LKB1/AMPK/mTOR axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4662.

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

Purpose: The main objectives of this exploratory analysis were to identify biomarkers in blood associated with clinical benefit from bevacizumab, and to assess CAF changes related to nephrectomy (Nx) in mRCC. Methods: Plasma samples were obtained from 44 pts participating in a frontline presurgical study of bevacizumab in clear cell mRCC (22 pts treated with BEV and 22 pts with BEV and erlotinib) (Jonasch et al. J Clin Oncol 2009), before treatment (BL), after 8 weeks of therapy/pre-nephrectomy (Nx), after 16 weeks of therapy/postNx, and every 4 weeks thereafter. Multiplex bead suspension arrays and ELISA were used to measure concentrations of 58 CAFs including VEGF, VEGF-C, placental growth factor (PlGF), soluble CAIX (sCA9), hepatocyte growth factor (HGF), osteopontin (OPN), matrix metalloproteinase-9 (MMP9), transforming growth factor (TGF)b1, endoglin, type IV collagen (ColIV), and multiple chemokines and interleukins (IL). CAFs were correlated with progression-free survival (PFS), overall survival (OS), and best response in primary and metastatic disease (% relative to BL). Results: Of 58 CAFs available at BL, high (above median) concentrations of TRAIL and ColIV were associated with longer PFS (7.6 v 2.8 and 8.1 v 2.9 months [mos], respectively; both P=0.001) and OS (27.9 v 13.2 and 32.8 v 14.3 mos, respectively; P=0.023 and P=0.025). In addition to high TRAIL, low levels of VEGF and PlGF also showed a significant association with enhanced response both in primary disease and metastases (all P Conclusions: We identified TRAIL, ColIV, VEGF and PlGF as candidate CAFs associated with clinical benefit from BEV in clear cell mRCC. The combination of BEV with erlotinib resulted in distinct on treatment CAF changes. The integration of these data with CAF profiling studies in pts with non-metastatic RCC could help define CAF sets useful to assist in risk stratification of RCC pts and provide insights into the biology of RCC progression. Citation Format: Alper Yetil, Eric Jonasch, Xuemei Wang, Christopher Wood, Surena Matin, Hai T. Tran, Nizar M. Tannir, John V. Heymach, Amado J. Zurita. Plasma cytokine and angiogenic factor (CAF) profiling for identification of markers associated with clinical benefit from bevacizumab (BEV) and with primary tumor presence in a phase II presurgical study of metastatic renal cell carcinoma (mRCC) patients [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 4645. doi:10.1158/1538-7445.AM2013-4645

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

Mutant KRAS (mut-KRAS) is present in 17-25% of all human cancers, where it plays a critical role in driving cancer cell growth and resistance to therapy. Despite numerous attempts, there is still no effective therapy for mut-KRAS tumors. Understanding the signaling mechanisms activated by mut-KRAS and finding agents to inhibit mut-KRAS signaling are important unmet needs in cancer therapy today. The recently completed BATTLE-1 clinical trial, a prospective, multi-arm, biopsy-mandated, biomarker-driven, clinical trial in advanced refractory non-small cell lung cancer (NSCLC), found that mut-KRAS did not accurately predict patient outcome (progression-free survival) to targeted intervention. This finding contradicted published evidence for such a relationship from colon cancer and some previous NSCLC studies. We explored more specifically the nature of the KRAS mutations, which are primarily found at codons 12 and 13, where different base substitutions lead to alternate amino acid (aa) substitutions. NSCLC has a much higher proportion of mut-KRAS G12C(cysteine) aa substitutions (47%) due to carcinogens in tobacco smoke, and only 15% mut-KRAS have G12D(aspartate). These data contrast those in other solid tumors, such as colon or pancreas, which predominantly manifest mut-KRAS G12D (50%) and only 9% mut-KRAS G12C. In a subset analysis of the BATTLE-1 data, we showed significantly worse progression-free survival in patients with mut-KRAS G12C, versus other mut-KRAS including G12D (p=.041) and who were treated with erlotinib, vandetanib or sorafenib. In a panel of NSCLC cell lines with known mut-KRAS aa substitutions to identify pathways activated by the different mut-KRAS genotypes, we found that mut-KRAS G12D activates both PI-3-K and MEK signaling, while mut-KRAS G12C does not and alternatively activates PKCζ and RAL signaling. This finding was confirmed in immortalized human bronchial epithelial (HBEC) cells stably transfected with wt-KRAS or different forms of mut-KRAS. Our molecular modeling studies show that the different conformation imposed by mut-KRAS G12C could lead to altered association with downstream signaling transducers, compared to mut-KRAS G12D. The significance of the findings for developing mut-KRAS therapies is profound, since it suggests that not all mut-KRAS may be addictive; and that different combinations of inhibitors of downstream signaling may be needed for different mut-KRAS. 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 955. doi:10.1158/1538-7445.AM2011-955

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