Publications by authors named "Jill M Siegfried"

115 Publications

Anti-EGFR Fibronectin Bispecific Chemically Self-Assembling Nanorings (CSANs) Induce Potent T Cell-Mediated Antitumor Responses and Downregulation of EGFR Signaling and PD-1/PD-L1 Expression.

J Med Chem 2020 09 14;63(18):10235-10245. Epub 2020 Sep 14.

Department of Virology, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil.

Overexpression of the epidermal growth factor receptor (EGFR) on various cancers makes it an important target for cancer immunotherapy. We recently demonstrated that single-chain variable fragment-based bispecific chemically self-assembled nanorings (CSANs) can successfully modify T cell surfaces and function as prosthetic antigen receptors (PARs) allowing selective targeting of tumor antigens while incorporating a dissociation mechanism of the rings. Here, we report the generation of anti-EGFR fibronectin (FN3)-based PARs with high yield, rapid protein production, predicted low immunogenicity, and increased protein stability. We demonstrated the cytotoxicity of FN3-PARs successfully while evaluating FN3 affinities, CSAN valencies, and antigen expression levels. Using an orthotopic breast cancer model, we showed that FN3-PARs can suppress tumor growth with no adverse effects and FN3-PARs reduced immunosuppressive programmed cell death ligand-1 (PD-L1) expression by downregulating EGFR signaling. These results demonstrate the potential of FN3-PARs to direct selective T cell-targeted tumor killing and to enhance antitumor T cell efficacy by modulating the tumor microenvironment.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00489DOI Listing
September 2020

Prevention of Tobacco Carcinogen-Induced Lung Tumor Development by a Novel STAT3 Decoy Inhibitor.

Cancer Prev Res (Phila) 2020 Sep 12;13(9):735-746. Epub 2020 Jul 12.

Department of Pharmacology, Medical School, and Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.

The STAT3 pathway is frequently overactive in non-small cell lung cancer (NSCLC), an often fatal disease with known risk factors including tobacco and chemical exposures. Whether STAT3 can be downmodulated to delay or prevent development of lung cancer resulting from an environmental exposure has not been previously tested. A circular oligonucleotide STAT3 decoy (CS3D) was used to treat mice previously exposed to the tobacco carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CS3D contains a double-stranded STAT3 DNA response element sequence and interrupts STAT3 signaling by binding to STAT3 dimers, rendering them unable to initiate transcription at native STAT3 DNA binding sites. An intermittent course of CS3D decreased the development of airway preneoplasias by 42% at 1 week posttreatment, reduced the progression of preneoplasia to adenomas by 54% at 8 weeks posttreatment, and reduced the size and number of resulting lung tumors by 49.7% and 29.5%, respectively, at 20 weeks posttreatment. No toxicity was detected. A mutant cyclic oligonucleotide with no STAT3 binding ability was used as a control. Chemopreventive effects were independent of the KRAS mutational status of the tumors. In lungs harvested during and after the treatment course with CS3D, airway preneoplasias had reduced STAT3 signaling. Chemopreventive effects were accompanied by decreased VEGFA expression, ablated IL6, COX-2, and p-NF-κB, and decreased pulmonary M2 macrophages and myeloid-derived suppressor cells. Thus, downmodulation of STAT3 activity using a decoy molecule both reduced oncogenic signaling in the airway epithelium and favored a lung microenvironment with reduced immunosuppression.
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http://dx.doi.org/10.1158/1940-6207.CAPR-20-0033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485626PMC
September 2020

Inhibiting Pathways Predicted From a Steroid Hormone Gene Signature Yields Synergistic Antitumor Effects in NSCLC.

J Thorac Oncol 2020 01 10;15(1):62-79. Epub 2019 Oct 10.

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota; Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota; Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania. Electronic address:

Introduction: Mounting evidence supports a role for estrogen signaling in NSCLC progression. We previously reported a seven-gene signature that predicts prognosis in estrogen receptor β positive (ERβ+) NSCLC. The signature defines a network comprised of ER and human EGFR-2/3 (HER2/HER3) signaling.

Methods: We tested the efficacy of combining the pan-HER inhibitor, dacomitinib, with the estrogen antagonist, fulvestrant, in ERβ+ NSCLC models with differing genotypes. We assessed the potency of this combination on xenograft growth and survival of host mice, and the ability to reverse the gene signature associated with poor outcome.

Results: Synergy was observed between dacomitinib and fulvestrant in three human ERβ+ NSCLC models: 201T (wild-type EGFR), A549 (KRAS mutant), and HCC827 (EGFR 19 deletion) with combination indices of 0.1-0.6. The combination, but not single agents, completely reversed the gene signature associated with poor prognosis in a mechanism that is largely mediated by activator protein 1 downregulation. In vivo, the combination also induced tumor regression and reversed the gene signature. In HCC827 xenografts treated with the combination, survival of mice was prolonged after therapy discontinuation, tumors that recurred were less aggressive, and two mechanisms of HER inhibitor resistance involving c-Met activation and PTEN loss were blocked.

Conclusions: The combination of an ER blocker and a pan-HER inhibitor provides synergistic efficacy in different models of ERβ+ NSCLC. Our data support the use of this combination clinically, considering its ability to induce potent antitumor effects and produce a gene signature that predicts better clinical outcomes.
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http://dx.doi.org/10.1016/j.jtho.2019.09.195DOI Listing
January 2020

Lung Adenocarcinoma Syndecan-2 Potentiates Cell Invasiveness.

Am J Respir Cell Mol Biol 2019 06;60(6):659-666

1 Division of Pulmonary and Critical Care Medicine, and.

Altered expression of syndecan-2 (SDC2), a heparan sulfate proteoglycan, has been associated with diverse types of human cancers. However, the mechanisms by which SDC2 may contribute to the pathobiology of lung adenocarcinoma have not been previously explored. SDC2 levels were measured in human lung adenocarcinoma samples and lung cancer tissue microarrays using immunohistochemistry and real-time PCR. To understand the role of SDC2 , SDC2 was silenced or overexpressed in A549 lung adenocarcinoma cells. The invasive capacity of cells was assessed using Matrigel invasion assays and measuring matrix metalloproteinase (MMP) 9 expression. Finally, we assessed tumor growth and metastasis of SDC2-deficient A549 cells in a xenograft tumor model. SDC2 expression was upregulated in malignant epithelial cells and macrophages obtained from human lung adenocarcinomas. Silencing of SDC2 decreased MMP9 expression and attenuated the invasive capacity of A549 lung adenocarcinoma cells. The inhibitory effect of SDC2 silencing on MMP9 expression and cell invasion was reversed by overexpression of MMP9 and syntenin-1. SDC2 silencing attenuated NF-κB p65 subunit nuclear translocation and its binding to the MMP9 promoter, which were restored by overexpression of syntenin-1. SDC2 silencing reduced tumor mass volume and metastasis. These findings suggest that SDC2 plays an important role in the invasive properties of lung adenocarcinoma cells and that its effects are mediated by syntenin-1. Thus, inhibiting SDC2 expression or activity could serve as a potential therapeutic target to treat lung adenocarcinoma.
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http://dx.doi.org/10.1165/rcmb.2018-0118OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543747PMC
June 2019

Status of Agents Targeting the HGF/c-Met Axis in Lung Cancer.

Cancers (Basel) 2018 Aug 21;10(9). Epub 2018 Aug 21.

Department of Pharmacology and Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

Hepatocyte growth factor (HGF) is the ligand for the tyrosine kinase receptor c-Met (Mesenchymal Epithelial Transition Factor also known as Hepatocyte Growth Factor Receptor, HGFR), a receptor with expression throughout epithelial and endothelial cell types. Activation of c-Met enhances cell proliferation, invasion, survival, angiogenesis, and motility. The c-Met pathway also stimulates tissue repair in normal cells. A body of past research shows that increased levels of HGF and/or overexpression of c-Met are associated with poor prognosis in several solid tumors, including lung cancer, as well as cancers of the head and neck, gastro-intestinal tract, breast, ovary and cervix. The HGF/c-Met signaling network is complex; both ligand-dependent and ligand-independent signaling occur. This article will provide an update on signaling through the HGF/c-Met axis, the mechanism of action of HGF/c-Met inhibitors, the lung cancer patient populations most likely to benefit, and possible mechanisms of resistance to these inhibitors. Although c-Met as a target in non-small cell lung cancer (NSCLC) showed promise based on preclinical data, clinical responses in NSCLC patients have been disappointing in the absence of mutation or gene amplification. New therapeutics that selectively target c-Met or HGF, or that target c-Met and a wider spectrum of interacting tyrosine kinases, will be discussed.
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http://dx.doi.org/10.3390/cancers10090280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162713PMC
August 2018

Randomized phase II study of fulvestrant and erlotinib compared with erlotinib alone in patients with advanced or metastatic non-small cell lung cancer.

Lung Cancer 2018 09 22;123:91-98. Epub 2018 Jun 22.

David Geffen School of Medicine at UCLA and Jonsson Comprehensive Cancer Center, 2825 Santa Monica Blvd, Suite 200, Santa Monica, CA, USA.

Objectives: This open-label, randomized phase II trial evaluated antitumor efficacy of an antiestrogen, fulvestrant, in combination with human epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in advanced non-small cell lung cancer (NSCLC) patients.

Materials And Methods: Patients with advanced or metastatic NSCLC, ECOG 0-2, previous chemotherapy unless patient refusal, and no prior EGFR-directed therapy were randomized 2:1 to erlotinib 150 mg oral daily plus 500 mg intramuscular fulvestrant on day 1, 15, 29 and every 28 days thereafter or erlotinib alone 150 mg oral daily. The primary end point was objective response rate (ORR); secondary endpoints included progression free survival (PFS) and overall survival (OS).

Results: Among 106 randomized patients, 100 received at least one dose of study drug. ORR was 16.4% (11 of 67 patients) for the combination versus 12.1% (4 of 33 patients) for erlotinib (p = 0.77). PFS median 3.5 versus 1.9 months [HR = 0.86, 95% CI (0.52-1.43), p = 0.29] and OS median 9.5 versus 5.8 months [HR = 0.92, 95% CI (0.57-1.48), p = 0.74] numerically favored the combination. In an unplanned subset analysis, among EGFR wild type patients (n = 51), but not EGFR mutant patients (n = 17), median PFS was 3.5 versus 1.7 months [HR = 0.35, 95% CI (0.14-0.86), p = 0.02] and OS was 6.2 versus 5.2 months [HR = 0.72, 95% CI (0.35-1.48), p = 0.37] for combined therapy versus erlotinib, respectively. Notably, EGFR WT patients were more likely to be hormone receptor-positive (either estrogen receptor α- and/or progesterone receptor-positive) compared to EGFR mutant patients (50% versus 9.1%, respectively) (p = 0.03). Treatment was well tolerated with predominant grade 1-2 dermatologic and gastrointestinal adverse effects.

Conclusion: Addition of fulvestrant to erlotinib was well tolerated, with increased activity noted among EGFR wild type patients compared to erlotinib alone, albeit in an unplanned subset analysis.
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http://dx.doi.org/10.1016/j.lungcan.2018.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6118115PMC
September 2018

STAT3 Cyclic Decoy Demonstrates Robust Antitumor Effects in Non-Small Cell Lung Cancer.

Mol Cancer Ther 2018 09 11;17(9):1917-1926. Epub 2018 Jun 11.

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.

Constitutively activated STAT3 plays a critical role in non-small cell lung carcinoma (NSCLC) progression by mediating proliferation and survival. STAT3 activation in normal cells is transient, making it an attractive target for NSCLC therapy. The therapeutic potential of blocking STAT3 in NSCLC was assessed utilizing a decoy approach by ligating a double-stranded 15-mer oligonucleotide that corresponds to the STAT3 response element of STAT3-target genes, to produce a cyclic STAT3 decoy (CS3D). The decoy was evaluated using NSCLC cells containing either wild-type EGFR (201T) or mutant EGFR with an additional EGFRi resistance mutation (H1975). These cells are resistant to EGFR inhibitors and require an alternate therapeutic approach. CS3D activity was compared with an inactive cyclic control oligonucleotide (CS3M) that differs by a single base pair, rendering it unable to bind to STAT3 protein. Transfection of 0.3 μmol/L of CS3D caused a 50% inhibition in proliferation in 201T and H1975 cells, relative to CS3M, and a 2-fold increase in apoptotic cells. Toxicity was minimal in normal cells. CS3D treatment caused a significant reduction of mRNA and protein expression of the STAT3 target gene c-Myc and inhibited colony formation by 70%. The active decoy decreased the nuclear pool of STAT3 compared with the mutant. In a xenograft model, treatments with CS3D (5 mg/kg) caused a potent 96.5% and 81.7% reduction in tumor growth in 201T ( < 0.007) and H1975 models ( < 0.0001), respectively, and reduced c-Myc and p-STAT3 proteins. Targeting STAT3 with the cyclic decoy could be an effective therapeutic strategy for NSCLC. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-1194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125196PMC
September 2018

Gene Promoter Hypermethylation Detected in Sputum Predicts FEV Decline and All-Cause Mortality in Smokers.

Am J Respir Crit Care Med 2018 07;198(2):187-196

1 Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico.

Rationale: Gene promoter hypermethylation detected in sputum assesses the extent of field cancerization and predicts lung cancer (LC) risk in ever-smokers. A rapid decline of FEV is a major driver for development of airway obstruction.

Objectives: To assess the effects of methylation of 12 genes on FEV decline and of FEV decline on subsequent LC incidence using two independent, longitudinal cohorts (i.e., LSC [Lovelace Smokers Cohort] and PLuSS [Pittsburgh Lung Screening Study]).

Methods: Gene methylation was measured in sputum using two-stage nested methylation-specific PCR. The linear mixed effects model was used to assess the effects of studied variables on FEV decline.

Measurements And Main Results: A dose-dependent relationship between number of genes methylated and FEV decline was identified, with smokers with three or more methylated genes having 27.8% and 10.3% faster FEV decline than smokers with zero to two methylated genes in the LSC and PLuSS cohort, respectively (all P < 0.01). High methylation in sputum was associated with a shorter latency for LC incidence (log-rank P = 0.0048) and worse all-cause mortality (log-rank P < 0.0001). Smokers with subsequent LC incidence had a more rapid annual decline of FEV (by 5.2 ml, P = 0.038) than smoker control subjects.

Conclusions: Gene methylation detected in sputum predicted FEV decline, LC incidence, and all-cause mortality in smokers. Rapid FEV decline may be a risk factor for LC incidence in smokers, which may explain a greater prevalence of airway obstruction seen in patients with LC.
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http://dx.doi.org/10.1164/rccm.201708-1659OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058990PMC
July 2018

Preclinical Evidence for Combined Use of Aromatase Inhibitors and NSAIDs as Preventive Agents of Tobacco-Induced Lung Cancer.

J Thorac Oncol 2018 03 9;13(3):399-412. Epub 2017 Dec 9.

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.

Introduction: A hormonal role in NSCLC development is well documented. We previously showed that the aromatase inhibitor (AI) anastrozole decreased development of tobacco carcinogen-induced lung tumors in a murine lung cancer prevention model and that aromatase and estrogen receptor were expressed in pulmonary inflammatory cells.

Methods: We utilized a tobacco carcinogen-induced lung tumor mouse model by treatment with 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK), to determine whether an AI combined with nonsteroidal anti-inflammatory drugs results in greater lung tumor prevention effects compared to single-agent treatment.

Results: Combination of anastrozole (0.1 mg/kg/d) with aspirin (25 mg/kg/d) after NNK exposure resulted in significantly fewer and smaller lung tumors than did single-agent treatments and was accompanied by maximum decreases in circulating β-estradiol (E2) and interleukin-6, tumor-infiltrating macrophages, and tumoral Ki67, phospho-mitogen-activated protein kinase, phospho-signal transducer and activator of transcription 3, and interleukin-17A expression. Preneoplasia arising after combination treatment showed the lowest Sox-2 expression, suggesting an inhibitory effect on proliferative capacity in the airways by blocking both E2 and inflammation. Anastrozole combined with ibuprofen instead of aspirin also showed enhanced antitumor effects. Moreover, male mice treated with NNK that received E2 in their drinking water showed greater levels of pulmonary macrophages and inflammatory markers than did the control, confirming an E2 effect on inflammation in the microenvironment.

Conclusions: Our results suggest a benefit to joint targeting of the estrogen and inflammatory pathways for NSCLC prevention. Combining AIs with nonsteroidal anti-inflammatory drugs reduces circulating E2, proinflammatory cytokines, and macrophage recruitment in the lung microenvironment after tobacco exposure. This strategy could be particularly effective in women who have underlying pulmonary inflammatory diseases.
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http://dx.doi.org/10.1016/j.jtho.2017.11.126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825271PMC
March 2018

Gene methylation biomarkers in sputum as a classifier for lung cancer risk.

Oncotarget 2017 Sep 15;8(38):63978-63985. Epub 2017 Jul 15.

Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, NM, USA.

CT screening for lung cancer reduces mortality, but will cost Medicare ∼2 billion dollars due in part to high false positive rates. Molecular biomarkers could augment current risk stratification used to select smokers for screening. Gene methylation in sputum reflects lung field cancerization that remains in lung cancer patients post-resection. This population was used in conjunction with cancer-free smokers to evaluate classification accuracy of a validated eight-gene methylation panel in sputum for cancer risk. Sputum from resected lung cancer patients (n=487) and smokers from Lovelace (n=1380) and PLuSS (n=718) cohorts was studied for methylation of an 8-gene panel. Area under a receiver operating characteristic curve was calculated to assess the prediction performance in logistic regressions with different sets of variables. The prevalence for methylation of all genes was significantly increased in the ECOG-ACRIN patients compared to cancer-free smokers as evident by elevated odds ratios that ranged from 1.6 to 8.9. The gene methylation panel showed lung cancer prediction accuracy of 82-86% and with addition of clinical variables improved to 87-90%. With sensitivity at 95%, specificity increased from 25% to 54% comparing clinical variables alone to their inclusion with methylation. The addition of methylation biomarkers to clinical variables would reduce false positive screens by ruling out one-third of smokers eligible for CT screening and could increase cancer detection rates through expanding risk assessment criteria.
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http://dx.doi.org/10.18632/oncotarget.19255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609978PMC
September 2017

Lung Endothelial MicroRNA-1 Regulates Tumor Growth and Angiogenesis.

Am J Respir Crit Care Med 2017 12;196(11):1443-1455

1 Section of Pulmonary, Critical Care, and Sleep Medicine and.

Rationale: Vascular endothelial growth factor down-regulates microRNA-1 (miR-1) in the lung endothelium, and endothelial cells play a critical role in tumor progression and angiogenesis.

Objectives: To examine the clinical significance of miR-1 in non-small cell lung cancer (NSCLC) and its specific role in tumor endothelium.

Methods: miR-1 levels were measured by Taqman assay. Endothelial cells were isolated by magnetic sorting. We used vascular endothelial cadherin promoter to create a vascular-specific miR-1 lentiviral vector and an inducible transgenic mouse. KRAS/Trp (KP) mice, lung-specific vascular endothelial growth factor transgenic mice, Lewis lung carcinoma xenografts, and primary endothelial cells were used to test the effects of miR-1.

Measurements And Main Results: In two cohorts of patients with NSCLC, miR-1 levels were lower in tumors than the cancer-free tissue. Tumor miR-1 levels correlated with the overall survival of patients with NSCLC. miR-1 levels were also lower in endothelial cells isolated from NSCLC tumors and tumor-bearing lungs of KP mouse model. We examined the significance of lower miR-1 levels by testing the effects of vascular-specific miR-1 overexpression. Vector-mediated delivery or transgenic overexpression of miR-1 in endothelial cells decreased tumor burden in KP mice, reduced the growth and vascularity of Lewis lung carcinoma xenografts, and decreased tracheal angiogenesis in vascular endothelial growth factor transgenic mice. In endothelial cells, miR-1 level was regulated through phosphoinositide 3-kinase and specifically controlled proliferation, de novo DNA synthesis, and ERK1/2 activation. Myeloproliferative leukemia oncogene was targeted by miR-1 in the lung endothelium and regulated tumor growth and angiogenesis.

Conclusions: Endothelial miR-1 is down-regulated in NSCLC tumors and controls tumor progression and angiogenesis.
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http://dx.doi.org/10.1164/rccm.201610-2157OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736970PMC
December 2017

Clinical Utility of Chromosomal Aneusomy in Individuals at High Risk of Lung Cancer.

J Thorac Oncol 2017 10 19;12(10):1512-1523. Epub 2017 Jun 19.

Department of Medicine, Division of Medical Oncology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado; Department of Pathology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Introduction: Low-dose computed tomography screening for lung cancer has a high false-positive rate with frequent discovery of indeterminate pulmonary nodules. Noninvasive biomarkers are needed to reduce false positives and improve risk stratification. A retrospective longitudinal evaluation was performed to assess chromosomal aneusomy in sputum by fluorescence in situ hybridization (CA-FISH) in four nested case-control studies.

Methods: Receiver operating characteristic analysis resulted in two grouped cohorts: a high-risk cohort (Colorado High-Risk Cohort and Colorado Nodule Cohort [68 case patients and 69 controls]) and a screening cohort (American College of Radiology Imaging Network/National Lung Screening Trial and Pittsburgh Lung Screening Study [97 case patients and 185 controls]). The CA-FISH assay was a four-target DNA panel encompassing the EGFR and v-myc avian myelocytomatosis viral oncogene homolog (MYC) genes, and the 5p15 and centromere 6 regions or the fibroblast growth factor 1 gene (FGFR1) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA). A four-category scale (normal, probably normal, probably abnormal, and abnormal) was applied. Sensitivity, specificity, and positive and negative likelihood ratios (LRs) (with 95% confidence intervals [CIs]) were estimated for each cohort.

Results: Sensitivity and specificity were, respectively, 0.67 (95% CI: 0.55-0.78) and 0.94 (95% CI: 0.85-0.98) for high-risk participants and 0.20 (95% CI: 0.13-0.30) and 0.84 (95% CI: 0.78-0.89) for screening participants. The positive and negative LRs were, respectively, 11.66 (95% CI: 4.44-30.63) and 0.34 (95% CI: 0.24-0.48) for high-risk participants and 1.36 (95% CI: 0.81-2.28) and 0.93 (95% CI: 0.83-1.05) for screening participants.

Conclusion: The high positive LR of sputum CA-FISH indicates that it could be a useful adjunct to low-dose computed tomography for lung cancer in high-risk settings. For screening, however, its low positive LR limits clinical utility. Prospective assessment of CA-FISH in the incidentally identified indeterminate nodule setting is ongoing in the Colorado Pulmonary Nodule Biomarker Trial.
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http://dx.doi.org/10.1016/j.jtho.2017.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647865PMC
October 2017

Phase 1/2 study of rilotumumab (AMG 102), a hepatocyte growth factor inhibitor, and erlotinib in patients with advanced non-small cell lung cancer.

Cancer 2017 Aug 4;123(15):2936-2944. Epub 2017 May 4.

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.

Background: Activation of the mesenchymal-epidermal transition factor (MET) tyrosine kinase and its ligand, hepatocyte growth factor (HGF), is implicated in resistance to epidermal growth factor receptor (EGFR) inhibitors. In this phase 1/2 trial, rilotumumab (an anti-HGF antibody) combined with erlotinib was evaluated in patients with metastatic, previously treated non-small cell lung cancer.

Methods: In phase 1, a dose de-escalation design was adopted with rilotumumab starting at 15 mg/kg intravenously every 3 weeks and oral erlotinib 150 mg daily. In phase 2, the disease control rate (DCR) (according to Response Evaluation Criteria in Solid Tumors) of the combination was evaluated using a Simon 2-stage design. The biomarkers examined included 10 plasma-circulating molecules associated with the EGFR and MET pathways.

Results: Without indications for de-escalation, the recommended phase 2 dose was dose level 0. Overall, 45 response-evaluable patients were enrolled (13 with squamous carcinoma, 32 with adenocarcinoma; 2 had confirmed EGFR mutations, 33 had confirmed wild-type [WT] EGFR, and 7 had KRAS mutations). The DCR for all patients was 60% (90% confidence interval [CI], 47.1%-71.3%). Median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 6.6 months (90% CI, 5.6-8.9 months). Among patients with WT EGFR, the DCR was 60.6% (90% CI, 46.3%-73.3%), median progression-free survival was 2.6 months (90% CI, 1.4-2.7 months), and median overall survival was 7.0 months (90% CI, 5.6-13.4 months). Elevated baseline levels of neuregulin 1 were associated with longer progression-free survival (hazard ratio, 0.41; 95% CI, 0.19-0.87), whereas elevated amphiregulin levels were associated with more rapid progression (hazard ratio, 2.14; 95% CI, 1.48-3.08).

Conclusions: Combined rilotumumab and erlotinib had an acceptable safety profile, and the DCR met the prespecified criteria for success. In the EGFR WT group, the DCR exceeded published reports for erlotinib alone. High circulating levels of neuregulin 1 may indicate sensitivity to this combination. Cancer 2017;123:2936-44. © 2017 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.30717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517339PMC
August 2017

Interaction between the estrogen receptor and fibroblast growth factor receptor pathways in non-small cell lung cancer.

Oncotarget 2017 Apr;8(15):24063-24076

Department of Pharmacology & Chemical Biology and University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA, USA.

The estrogen receptor (ER) promotes non-small cell lung cancer (NSCLC) proliferation. Since fibroblast growth factors (FGFs) are known regulators of stem cell markers in ER positive breast cancer, we investigated whether a link between the ER, FGFs, and stem cell markers exists in NSCLC. In lung preneoplasias and adenomas of tobacco carcinogen exposed mice, the anti-estrogen fulvestrant and/or the aromatase inhibitor anastrozole blocked FGF2 and FGF9 secretion, and reduced expression of the stem cell markers SOX2 and nanog. Mice administered β-estradiol during carcinogen exposure showed increased FGF2, FGF9, SOX2, and Nanog expression in airway preneoplasias. In normal FGFR1 copy number NSCLC cell lines, multiple FGFR receptors were expressed and secreted several FGFs. β-estradiol caused enhanced FGF2 release, which was blocked by fulvestrant. Upon co-inhibition of ER and FGFRs using fulvestrant and the pan-FGFR inhibitor AZD4547, phosphorylation of FRS2, the FGFR docking protein, was maximally reduced, and enhanced anti-proliferative effects were observed. Combined AZD4547 and fulvestrant enhanced lung tumor xenograft growth inhibition and decreased Ki67 and stem cell marker expression. To verify a link between ERβ, the predominant ER in NSCLC, and FGFR signaling in patient tumors, mRNA analysis was performed comparing high versus low ERβ expressing tumors. The top differentially expressed genes in high ERβ tumors involved FGF signaling and human embryonic stem cell pluripotency. These results suggest interaction between the ER and FGFR pathways in NSCLC promotes a stem-like state. Combined FGFR and ER inhibition may increase the efficacy of FGFR inhibitors for NSCLC patients lacking FGFR genetic alterations.
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http://dx.doi.org/10.18632/oncotarget.16030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5421827PMC
April 2017

Myeloid STAT3 Promotes Lung Tumorigenesis by Transforming Tumor Immunosurveillance into Tumor-Promoting Inflammation.

Cancer Immunol Res 2017 03 20;5(3):257-268. Epub 2017 Jan 20.

University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania.

One of the most fundamental and challenging questions in the cancer field is how immunity in patients with cancer is transformed from tumor immunosurveillance to tumor-promoting inflammation. Here, we identify the transcription factor STAT3 as the culprit responsible for this pathogenic event in lung cancer development. We found that antitumor type 1 CD4 T-helper (Th1) cells and CD8 T cells were directly counter balanced in lung cancer development with tumor-promoting myeloid-derived suppressor cells (MDSCs) and suppressive macrophages, and that activation of STAT3 in MDSCs and macrophages promoted tumorigenesis through pulmonary recruitment and increased resistance of suppressive cells to CD8 T cells, enhancement of cytotoxicity toward CD4 and CD8 T cells, induction of regulatory T cell (Treg), inhibition of dendritic cells (DC), and polarization of macrophages toward the M2 phenotype. The deletion of myeloid STAT3 boosted antitumor immunity and suppressed lung tumorigenesis. These findings increase our understanding of immune programming in lung tumorigenesis and provide a mechanistic basis for developing STAT3-based immunotherapy against this and other solid tumors. .
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http://dx.doi.org/10.1158/2326-6066.CIR-16-0073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334370PMC
March 2017

A prospective and retrospective analysis of smoking behavior changes in ever smokers with high risk for lung cancer from New Mexico and Pennsylvania.

Int J Mol Epidemiol Genet 2016 1;7(2):95-104. Epub 2016 Jun 1.

Lung Cancer Program, Lovelace Respiratory Research Institute Albuquerque, NM.

Cigarette smoking is the leading preventable cause of death worldwide. The aim of this study is to conduct a prospective and retrospective analysis of smoking behavior changes in the Lovelace Smokers Cohort (LSC) and the Pittsburgh Lung Screening Study cohort (PLuSS). Area under the curve (AUC) for risk models predicting relapse based on demographic, smoking, and relevant clinical variables was 0.93 and 0.79 in LSC and PLuSS, respectively. The models for making a quit attempt had limited prediction ability in both cohorts (AUC≤0.62). We identified an ethnic disparity in adverse smoking behavior change that Hispanic smokers were less likely to make a quit attempt and were more likely to relapse after a quit attempt compared to non-Hispanic Whites. SNPs at 15q25 and 11p14 loci were associated with risk for smoking relapse in the LSC. Rs6495308 at 15q25 has a large difference in minor allele frequency between non-Hispanic Whites and Hispanics (0.46 versus 0.23, P<0.0001) and was associated with risk for ever relapse at same magnitude between the two ethnic groups (OR=1.36, 95% CI=1.10 to 1.67 versus 1.59, 95% CI=1.00 to 2.53, P=0.81). In summary, the risk prediction model established in LSC and PLuSS provided an excellent to outstanding distinguishing for abstainers who will or will not relapse. The ethnic disparity in adverse smoking behavior between Hispanics and non-Hispanic Whites may be at least partially explained by the sequence variants at 15q25 locus that contains multiple nicotine acetylcholine receptors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4913225PMC
June 2016

ATM protein is deficient in over 40% of lung adenocarcinomas.

Oncotarget 2016 09;7(36):57714-57725

Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Lung cancer is the leading cause of cancer-related mortality in the USA and worldwide, and of the estimated 1.2 million new cases of lung cancer diagnosed every year, over 30% are lung adenocarcinomas. The backbone of 1st-line systemic therapy in the metastatic setting, in the absence of an actionable oncogenic driver, is platinum-based chemotherapy. ATM and ATR are DNA damage signaling kinases activated at DNA double-strand breaks (DSBs) and stalled and collapsed replication forks, respectively. ATM protein is lost in a number of cancer cell lines and ATR kinase inhibitors synergize with cisplatin to resolve xenograft models of ATM-deficient lung cancer. We therefore sought to determine the frequency of ATM loss in a tissue microarray (TMA) of lung adenocarcinoma. Here we report the validation of a commercial antibody (ab32420) for the identification of ATM by immunohistochemistry and estimate that 61 of 147 (41%, 95% CI 34%-50%) cases of lung adenocarcinoma are negative for ATM protein expression. As a positive control for ATM staining, nuclear ATM protein was identified in stroma and immune infiltrate in all evaluable cases. ATM loss in lung adenocarcinoma was not associated with overall survival. However, our preclinical findings in ATM-deficient cell lines suggest that ATM could be a predictive biomarker for synergy of an ATR kinase inhibitor with standard-of-care cisplatin. This could improve clinical outcome in 100,000's of patients with ATM-deficient lung adenocarcinoma every year.
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http://dx.doi.org/10.18632/oncotarget.9757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5295384PMC
September 2016

KRAS and TP53 mutations in bronchoscopy samples from former lung cancer patients.

Mol Carcinog 2017 02 6;56(2):381-388. Epub 2016 Jun 6.

Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania.

Mutations in the KRAS and TP53 genes have been found frequently in lung tumors and specimens from individuals at high risk for lung cancer and have been suggested as predictive markers for lung cancer. In order to assess the prognostic value of these two genes' mutations in lung cancer recurrence, we analyzed mutations in codon 12 of the KRAS gene and in hotspot codons of the TP53 gene in 176 bronchial biopsies obtained from 77 former lung cancer patients. Forty-seven patients (61.0%) showed mutations, including 35/77 (45.5%) in the KRAS gene and 25/77 (32.5%) in the TP53 gene, among them 13/77 (16.9%) had mutations in both genes. When grouped according to past or current smoking status, a higher proportion of current smokers showed mutations, in particular those in the TP53 gene (P = 0.07), compared with ex-smokers. These mutations were found in both abnormal lesions (8/20 or 40%) and histologically normal tissues (70/156 or 44.9%) (P = 0.812). They consisted primarily of G to A transition and G to T transversion in both the KRAS (41/56 or 73.2%) and TP53 (24/34 or 70.6%) genes, consistent with mutations found in lung tumors of smoking lung cancer patients. Overall, recurrence-free survival (RFS) among all subjects could be explained by age at diagnosis, tumor stage, tumor subtype, and smoking (P < 0.05, Cox proportional hazard). Therefore, KRAS and TP53 mutations were frequently detected in bronchial tissues of former lung cancer patients. However, the presence of mutation of bronchial biopsies was not significantly associated with a shorter RFS time. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/mc.22501DOI Listing
February 2017

Expression of PAM50 Genes in Lung Cancer: Evidence that Interactions between Hormone Receptors and HER2/HER3 Contribute to Poor Outcome.

Neoplasia 2015 Nov;17(11):817-25

Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213; University of Pittsburgh Cancer Institute, University of Pittsburgh, Pittsburgh, PA 15213. Electronic address:

Non-small cell lung cancers (NSCLCs) frequently express estrogen receptor (ER) β, and estrogen signaling is active in many lung tumors. We investigated the ability of genes contained in the prediction analysis of microarray 50 (PAM50) breast cancer risk predictor gene signature to provide prognostic information in NSCLC. Supervised principal component analysis of mRNA expression data was used to evaluate the ability of the PAM50 panel to provide prognostic information in a stage I NSCLC cohort, in an all-stage NSCLC cohort, and in The Cancer Genome Atlas data. Immunohistochemistry was used to determine status of ERβ and other proteins in lung tumor tissue. Associations with prognosis were observed in the stage I cohort. Cross-validation identified seven genes that, when analyzed together, consistently showed survival associations. In pathway analysis, the seven-gene panel described one network containing the ER and progesterone receptor, as well as human epidermal growth factor receptor (HER)2/HER3 and neuregulin-1. NSCLC cases also showed a significant association between ERβ and HER2 protein expression. Cases positive for HER2 expression were more likely to express HER3, and ERβ-positive cases were less likely to be both HER2 and HER3 negative. Prognostic ability of genes in the PAM50 panel was verified in an ERβ-positive cohort representing all NSCLC stages. In The Cancer Genome Atlas data sets, the PAM50 gene set was prognostic in both adenocarcinoma and squamous cell carcinoma, whereas the seven-gene panel was prognostic only in squamous cell carcinoma. Genes in the PAM50 panel, including those linking ER and HER2, identify lung cancer patients at risk for poor outcome, especially among ERβ-positive cases and squamous cell carcinoma.
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http://dx.doi.org/10.1016/j.neo.2015.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681883PMC
November 2015

Functional analyses of ATM, ATR and Fanconi anemia proteins in lung carcinoma : ATM, ATR and FA in lung carcinoma.

BMC Cancer 2015 Oct 5;15:649. Epub 2015 Oct 5.

Department of Radiation Oncology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.

Background: ATM and ATR are kinases implicated in a myriad of DNA-damage responses. ATM kinase inhibition radiosensitizes cells and selectively kills cells with Fanconi anemia (FA) gene mutations. ATR kinase inhibition sensitizes cells to agents that induce replication stress and selectively kills cells with ATM and TP53 mutations. ATM mutations and FANCF promoter-methylation are reported in lung carcinomas.

Methods: We undertook functional analyses of ATM, ATR, Chk1 and FA proteins in lung cancer cell lines. We included Calu6 that is reported to be FANCL-deficient. In addition, the cancer genome atlas (TCGA) database was interrogated for alterations in: 1) ATM, MRE11A, RAD50 and NBN; 2) ATR, ATRIP and TOPBP1; and 3) 15 FA genes.

Results: No defects in ATM, ATR or Chk1 kinase activation, or FANCD2 monoubiquitination were identified in the lung cancer cell lines examined, including Calu6, and major alterations in these pathways were not identified in the TCGA database. Cell lines were radiosensitized by ATM kinase inhibitor KU60019, but no cell killing by ATM kinase inhibitor alone was observed. While no synergy between gemcitabine or carboplatin and ATR kinase inhibitor ETP-46464 was observed, synergy between gemcitabine and Chk1 kinase inhibitor UCN-01 was observed in 54 T, 201 T and H460, and synergy between carboplatin and Chk1 kinase inhibitor was identified in 201 T and 239 T. No interactions between ATM, ATR and FA activation were observed by either ATM or ATR kinase inhibition in the lung cancer cell lines.

Conclusions: Analyses of ATM serine 1981 and Chk1 serine 345 phosphorylation, and FANCD2 monoubiquitination revealed that ATM and ATR kinase activation and FA pathway signaling are intact in the lung cancer cell lines examined. As such, these posttranslational modifications may have utility as biomarkers for the integrity of DNA damage signaling pathways in lung cancer. Different sensitization profiles between gemcitabine and carboplatin and ATR kinase inhibitor ETP-46464 and Chk1 kinase inhibitor UCN-01 were observed and this should be considered in the rationale for Phase I clinical trial design with ATR kinase inhibitors.
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http://dx.doi.org/10.1186/s12885-015-1649-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595318PMC
October 2015

Lung Cancer Risk Prediction Using Common SNPs Located in GWAS-Identified Susceptibility Regions.

J Thorac Oncol 2015 Nov;10(11):1538-45

Departments of *Epidemiology and †Biostatistics, Graduate School of Public Health, Pittsburgh, Pennsylvania; Departments of ‡Medicine, §Radiology, and ‖Cardiothoracic Surgery, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; and ¶Department of Pharmacology, School of Medicine, University of Minnesota, Minneapolis, Minnesota.

Introduction: Genome-wide association studies (GWAS) have consistently identified specific lung cancer susceptibility regions. We evaluated the lung cancer-predictive performance of single-nucleotide polymorphisms (SNPs) in these regions.

Methods: Lung cancer cases (N = 778) and controls (N = 1166) were genotyped for 77 SNPs located in GWAS-identified lung cancer susceptibility regions. Variable selection and model development used stepwise logistic regression and decision-tree analyses. In a subset nested in the Pittsburgh Lung Screening Study, change in area under the receiver operator characteristic curve and net reclassification improvement were used to compare predictions made by risk factor models with and without genetic variables.

Results: Variable selection and model development kept two SNPs in each of three GWAS regions, rs2736100 and rs7727912 in 5p15.33, rs805297 and rs1802127 in 6p21.33, and rs8034191 and rs12440014 in 15q25.1. The ratio of cases to controls was three times higher among subjects with a high-risk genotype in every one as opposed to none of the three GWAS regions (odds ratio, 3.14; 95% confidence interval, 2.02-4.88; adjusted for sex, age, and pack-years). Adding a three-level classified count of GWAS regions with high-risk genotypes to an age and smoking risk factor-only model improved lung cancer prediction by a small amount: area under the receiver operator characteristic curve, 0.725 versus 0.717 (p = 0.056); overall net reclassification improvement was 0.052 across low-, intermediate-, and high- 6-year lung cancer risk categories (<3.0%, 3.0%-4.9%, ≥ 5.0%).

Conclusion: Specifying genotypes for SNPs in three GWAS-identified susceptibility regions improved lung cancer prediction, but probably by an extent too small to affect disease control practice.
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http://dx.doi.org/10.1097/JTO.0000000000000666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4636453PMC
November 2015

Epigenetic Repression of CCDC37 and MAP1B Links Chronic Obstructive Pulmonary Disease to Lung Cancer.

J Thorac Oncol 2015 Aug;10(8):1181-8

*Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico; †Department of Epidemiology, University of Pittsburgh, Pittsburgh, Pennsylvania; ‡Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota; and §COPD Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico.

Introduction: Lung cancer and chronic obstructive pulmonary disease (COPD) share environmental risk factors. COPD also increases the risk of lung cancer; however, the molecular mechanisms are unclear.

Methods: An epigenome-wide association study of lung tumors and cancer-free lung tissue (CFLT) pairs from non-small-cell lung cancer cases with (n = 18) or without (n = 17) COPD was conducted using the HumanMethylation450 beadchip (HM450K). COPD-associated methylation of top-ranked genes was confirmed in a larger sample set, independently validated, and their potential as sputum-based biomarkers was investigated.

Results: Methylation of CCDC37 and MAP1B was more prevalent in lung tumors from COPD than non-COPD cases [54 of 71 (76%) versus 20 of 46 (43%), p = 0.0013] and [48 of 71 (68%) versus 17 of 46 (37%), p = 0.0035], respectively, after adjustment for age, sex, smoking status, and tumor histology. HM450K probes across CCDC37 and MAP1B promoters showed higher methylation in tumors than CFLT with the highest methylation seen in tumors from COPD cases (p < 0.05). These results were independently validated using The Cancer Genome Atlas data. CCDC37 methylation was more prevalent in sputum from COPD than non-COPD smokers (p < 0.005) from two cohorts. CCDC37 and MAP1B expression was dramatically repressed in tumors and CFLT from COPD than non-COPD cases, p less than 0.02.

Conclusions: The reduced expression of CCDC37 and MAP1B associated with COPD likely predisposes these genes to methylation that in turn, may contribute to lung cancer.
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http://dx.doi.org/10.1097/JTO.0000000000000592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511946PMC
August 2015

15q12 variants, sputum gene promoter hypermethylation, and lung cancer risk: a GWAS in smokers.

J Natl Cancer Inst 2015 Feb 23;107(5). Epub 2015 Feb 23.

: Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, NM (SL, YL, CLT, MAP, RPW, KCD, XZ, EAB, YT, SAB); Department of Epidemiology, Graduate School of Public Health (JLW) and Department of Medicine (TN, MR), University of Pittsburgh, Pittsburgh, PA; Center for Genomic Medicine, Department of Community and Family Medicine, Geisel School of Medicine, Dartmouth College, Hanover, NH (YH, CIA); Department of Preventive Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA (CKE, DVDB, WJG, FDG); Department of Pharmacology & Chemical Biology, Hillman Cancer Center of the University of Pittsburgh Medical Center, Pittsburgh, PA (ALGD, JMS); Department of Internal Medicine, University of New Mexico, Albuquerque, NM (REC, CAS); Department of Pharmacology, University of Minnesota, Minneapolis, MN (JMS).

Background: Lung cancer is the leading cause of cancer-related mortality worldwide. Detection of promoter hypermethylation of tumor suppressor genes in exfoliated cells from the lung provides an assessment of field cancerization that in turn predicts lung cancer. The identification of genetic determinants for this validated cancer biomarker should provide novel insights into mechanisms underlying epigenetic reprogramming during lung carcinogenesis.

Methods: A genome-wide association study using generalized estimating equations and logistic regression models was conducted in two geographically independent smoker cohorts to identify loci affecting the propensity for cancer-related gene methylation that was assessed by a 12-gene panel interrogated in sputum. All statistical tests were two-sided.

Results: Two single nucleotide polymorphisms (SNPs) at 15q12 (rs73371737 and rs7179575) that drove gene methylation were discovered and replicated with rs73371737 reaching genome-wide significance (P = 3.3×10(-8)). A haplotype carrying risk alleles from the two 15q12 SNPs conferred 57% increased risk for gene methylation (P = 2.5×10(-9)). Rs73371737 reduced GABRB3 expression in lung cells and increased risk for smoking-induced chronic mucous hypersecretion. Furthermore, subjects with variant homozygote of rs73371737 had a two-fold increase in risk for lung cancer (P = .0043). Pathway analysis identified DNA double-strand break repair by homologous recombination (DSBR-HR) as a major pathway affecting susceptibility for gene methylation that was validated by measuring chromatid breaks in lymphocytes challenged by bleomycin.

Conclusions: A functional 15q12 variant was identified as a risk factor for gene methylation and lung cancer. The associations could be mediated by GABAergic signaling that drives the smoking-induced mucous cell metaplasia. Our findings also substantiate DSBR-HR as a critical pathway driving epigenetic gene silencing.
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http://dx.doi.org/10.1093/jnci/djv035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555640PMC
February 2015

Incidence of head and neck squamous cell carcinoma among subjects at high risk of lung cancer: results from the Pittsburgh Lung Screening Study.

Cancer 2015 May 5;121(9):1431-5. Epub 2015 Jan 5.

School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.

Background: Earlier detection and diagnosis of head and neck squamous cell carcinoma (HNSCC) should lead to improved outcomes. However, to the authors' knowledge, no effective screening strategy has been identified to date. In the current study, the authors evaluated whether it would be useful to screen subjects targeted for lung cancer screening for HNSCC as well.

Methods: Medical records, death certificates, and cancer registry and questionnaire data were used to determine the number of observed incident HNSCC cases in the Pittsburgh Lung Screening Study (PLuSS), a cohort of current and former smokers aged ≥50 years with a ≥12.5 pack-year smoking history. The expected number of cases was estimated using stratum-specific incidence rates obtained from Surveillance, Epidemiology, and End Results data for 2000 through 2011. The standardized incidence ratio was calculated to examine the difference between the observed and expected number of cases.

Results: Of the 3587 at-risk participants in the PLuSS, 23 (0.64%) developed HNSCC over a total of 32,201 person-years of follow-up. This finding was significantly higher than expected based on incidence rates obtained from the Surveillance, Epidemiology, and End Results program (13.70 cases expected; standardized incidence ratio, 1.68 [95% confidence interval, 1.06-2.52]). The excess burden of HNSCC in the PLuSS was 28.9 cases per 100,000 person-years. Observed incident cases were significantly more often male, had started smoking at a younger age, smoked more per day, and had more pack-years of smoking than the rest of the PLuSS at-risk participants.

Conclusions: The results of the current study provide a rationale for offering head and neck cancer screening along with computed tomography screening for lung cancer. Randomized controlled trials that assess the effectiveness of adding examination of the head and neck area to lung cancer screening programs are warranted.
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http://dx.doi.org/10.1002/cncr.29189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409462PMC
May 2015

Matrix metalloproteinase-19 promotes metastatic behavior in vitro and is associated with increased mortality in non-small cell lung cancer.

Am J Respir Crit Care Med 2014 Oct;190(7):780-90

1 Section of Pulmonary, Critical Care and Sleep Medicine, Yale School of Medicine, New Haven, Connecticut.

Rationale: Lung cancer is the leading cause of cancer death in both men and women in the United States and worldwide. Matrix metalloproteinases (MMPs) have been implicated in the development and progression of lung cancer, but their role in the molecular pathogenesis of lung cancer remains unclear. We have found that MMP19, a relatively novel member of the MMP family, is overexpressed in lung tumors when compared with control subjects.

Objectives: To test the hypothesis that MMP19 plays a significant role in the development and progression of non-small cell lung cancer (NSCLC).

Methods: We have analyzed lung cancer gene expression data, immunostained lung tumors for MMP19, and performed in vitro assays to test the effects of MMP19 in NSCLC cells.

Measurements And Main Results: We found that MMP19 gene and protein expression is increased in lung cancer tumors compared with adjacent and histologically normal lung tissues. In three independent datasets, increased MMP19 gene expression conferred a poorer prognosis in NSCLC. In vitro, we found that overexpression of MMP19 promotes epithelial-mesenchymal transition, migration, and invasiveness in multiple NSCLC cell lines. Overexpression of MMP19 with a mutation at the catalytic site did not impair epithelial-mesenchymal transition or expression of prometastasis genes. We also found that miR-30 isoforms, a microRNA family predicted to target MMP19, is markedly down-regulated in human lung cancer and regulates MMP19 expression.

Conclusions: Taken together, these findings suggest that MMP19 is associated with the development and progression of NSCLC and may be a potential biomarker of disease severity and outcome.
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http://dx.doi.org/10.1164/rccm.201310-1903OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299607PMC
October 2014

Inherited variation in the ATP-binding cassette transporter ABCB1 and survival after chemotherapy for stage III-IV lung cancer.

J Thorac Oncol 2014 Sep;9(9):1264-71

*University of Pittsburgh Cancer Institute and †Department of Epidemiology, University of Pittsburgh Graduate School of Public Health; ‡Division of Hematology/Oncology, Department of Medicine, University of Pittsburgh School of Medicine; §Department of Medicine, University of Pittsburgh School of Medicine; ‖Department of Cardiothoracic Surgery, University of Pittsburgh School of Medicine; ¶Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA.

Background: The ATP-binding cassette transporter gene ABCB1 and the glutathione S-transferase gene GSTP1 code for a multidrug resistance protein and for a detoxifying phase II metabolic enzyme, respectively, with substrate specificities that include chemotherapy drugs often used to treat lung cancer.

Methods: We genotyped 11 ABCB1 and eight GSTP1 single nucleotide polymorphisms (SNPs) in 698 white lung cancer patients (all current or former cigarette smokers) and used log-rank test statistics and proportional hazards regression to evaluate associations between SNP genotype and survival.

Results: Using data from all 698 cases, one SNP in ABCB1 (rs2235013) was statistically significantly associated with overall survival (p = 0.038, log-rank test). Chemotherapy and stage jointly (p = 0.025) significantly modified the association between rs2235013 and survival, with statistically significant (p = 0.013, log-rank test) association observed in the subgroup of stage III to IV lung cancer patients who received chemotherapy as part of their first course of treatment (n = 160; 93.1% nonsmall cell). Patients who inherited the minor T allele at ABCB1 rs2235013 experienced better overall survival and recurrence-free survival (hazard ratio, per minor T allele, [95% confidence interval]: 0.66 [0.49-0.90] and 0.55 [0.31-0.95], respectively; adjusted for year of diagnosis, sex, age at diagnosis, cigarette pack years, and stage). In addition, in the advanced stage chemotherapy-treated subgroup, four ABCB1 SNPs (rs6949448, rs2235046, rs1128503, and rs10276036) in mutual high linkage disequilibrium with rs2235013 and an independent ABCB1 SNP (rs1045642) showed statistically significant association (p < 0.05) with survival.

Conclusions: Inherited variation in ABCB1 may affect survival specifically in advanced stage lung cancer patients who receive chemotherapy.
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http://dx.doi.org/10.1097/JTO.0000000000000262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134100PMC
September 2014

Validation of a blood protein signature for non-small cell lung cancer.

Clin Proteomics 2014 1;11(1):32. Epub 2014 Aug 1.

SomaLogic, Inc., Boulder, CO, USA.

Background: CT screening for lung cancer is effective in reducing mortality, but there are areas of concern, including a positive predictive value of 4% and development of interval cancers. A blood test that could manage these limitations would be useful, but development of such tests has been impaired by variations in blood collection that may lead to poor reproducibility across populations.

Results: Blood-based proteomic profiles were generated with SOMAscan technology, which measured 1033 proteins. First, preanalytic variability was evaluated with Sample Mapping Vectors (SMV), which are panels of proteins that detect confounders in protein levels related to sample collection. A subset of well collected serum samples not influenced by preanalytic variability was selected for discovery of lung cancer biomarkers. The impact of sample collection variation on these candidate markers was tested in the subset of samples with higher SMV scores so that the most robust markers could be used to create disease classifiers. The discovery sample set (n = 363) was from a multi-center study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smokers and benign pulmonary nodule controls. The analysis resulted in a 7-marker panel with an AUC of 0.85 for all cases (68% adenocarcinoma, 32% squamous) and an AUC of 0.93 for squamous cell carcinoma in particular. This panel was validated by making blinded predictions in two independent cohorts (n = 138 in the first validation and n = 135 in the second). The model was recalibrated for a panel format prior to unblinding the second cohort. The AUCs overall were 0.81 and 0.77, and for squamous cell tumors alone were 0.89 and 0.87. The estimated negative predictive value for a 15% disease prevalence was 93% overall and 99% for squamous lung tumors. The proteins in the classifier function in destruction of the extracellular matrix, metabolic homeostasis and inflammation.

Conclusions: Selecting biomarkers resistant to sample processing variation led to robust lung cancer biomarkers that performed consistently in independent validations. They form a sensitive signature for detection of lung cancer, especially squamous cell histology. This non-invasive test could be used to improve the positive predictive value of CT screening, with the potential to avoid invasive evaluation of nonmalignant pulmonary nodules.
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http://dx.doi.org/10.1186/1559-0275-11-32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123246PMC
August 2014

Co-targeting c-Met and COX-2 leads to enhanced inhibition of lung tumorigenesis in a murine model with heightened airway HGF.

J Thorac Oncol 2014 Sep;9(9):1285-93

Departments of *Pharmacology and Chemical Biology and †Biostatistics, University of Pittsburgh, Pittsburgh, Pennsylvania; and ‡Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.

Background: The hepatocyte growth factor (HGF)/c-Met pathway is often dysregulated in non-small-cell lung cancer (NSCLC). HGF activation of c-Met induces cyclooxygenase-2 (COX-2), resulting in downstream stimulation by prostaglandin E2 of additional pathways. Targeting both c-Met and COX-2 might lead to enhanced antitumor effects by blocking signaling upstream and downstream of c-Met.

Methods: Effects of crizotinib or celecoxib alone or in combination were tested in NSCLC cells in vitro and in mice transgenic for airway expression of human HGF.

Results: Proliferation and invasion of NSCLC cells treated with a combination of crizotinib and celecoxib were significantly lower compared with single treatments. Transgenic mice showed enhanced COX-2 expression localized to preneoplastic areas following exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, which was not present without carcinogen exposure. This shows that COX-2 activity is present during lung tumor development in a high HGF environment. After 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone treatment, a significant decrease in the number of lung tumors per animal was observed after 13-week treatments of crizotinib, celecoxib, or the combination compared with placebo (p < 0.001). With combination treatment, the number of tumors was also significantly lower than single agent treatment (p < 0.001). In the resulting lung tumors, P-c-Met, COX-2, prostaglandin E2, and P-MAPK were significantly downmodulated by combination treatment compared with single treatment. Expression of the epithelial-mesenchymal transition markers E-cadherin and snail was also modulated by combination treatment.

Conclusions: In the presence of high HGF, dual inhibition of c-Met and COX-2 may enhance antitumor effects. This combination may have clinical potential in NSCLCs with high HGF/c-Met expression or epithelial-mesenchymal transition phenotype.
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http://dx.doi.org/10.1097/JTO.0000000000000245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134355PMC
September 2014

Erlotinib, erlotinib-sulindac versus placebo: a randomized, double-blind, placebo-controlled window trial in operable head and neck cancer.

Clin Cancer Res 2014 Jun 11;20(12):3289-98. Epub 2014 Apr 11.

Authors' Affiliations: Division of Head and Neck Surgery, Department of Otolaryngology, Oregon Health and Science University, Portland, Oregon; Division of Hematology/Oncology, Department of Internal Medicine; Department of Pharmacology and Chemical Biology;Biostatistics Facility, University of Pittsburgh, University of Pittsburgh Cancer Institute; Departments of Otolaryngology and Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Otolaryngology, University of Kansas Medical Center, Kansas City, Kansas; Women's Health Integrated Research Center, Gynecologic Cancer Center of Excellence, Annandale, Virginia; Division of Hematology/Oncology, Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts; and Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota.

Purpose: The EGF receptor (EGFR) and COX2 pathways are upregulated in head and neck squamous cell carcinoma (HNSCC). Preclinical models indicate synergistic antitumor activity from dual blockade. We conducted a randomized, double-blind, placebo-controlled window trial of erlotinib, an EGFR inhibitor; erlotinib plus sulindac, a nonselective COX inhibitor; versus placebo.

Experimental Design: Patients with untreated, operable stage II-IVb HNSCC were randomized 5:5:3 to erlotinib, erlotinib-sulindac, or placebo. Tumor specimens were collected before and after seven to 14 days of treatment. The primary endpoint was change in Ki67 proliferation index. We hypothesized an ordering effect in Ki67 reduction: erlotinib-sulindac > erlotinib > placebo. We evaluated tissue microarrays by immunohistochemistry for pharmacodynamic modulation of EGFR and COX2 signaling intermediates.

Results: From 2005-2009, 47 patients were randomized for the target 39 evaluable patients. Thirty-four tumor pairs were of sufficient quality to assess biomarker modulation. Ki67 was significantly decreased by erlotinib or erlotinib-sulindac (omnibus comparison, two-sided Kruskal-Wallis, P = 0.04). Wilcoxon pairwise contrasts confirmed greater Ki67 effect in both erlotinib groups (erlotinib-sulindac vs. placebo, P = 0.043; erlotinib vs. placebo, P = 0.027). There was a significant trend in ordering of Ki67 reduction: erlotinib-sulindac > erlotinib > placebo (two-sided exact Jonckheere-Terpstra, P = 0.0185). Low baseline pSrc correlated with greater Ki67 reduction (R(2) = 0.312, P = 0.024).

Conclusions: Brief treatment with erlotinib significantly decreased proliferation in HNSCC, with additive effect from sulindac. Efficacy studies of dual EGFR-COX inhibition are justified. pSrc is a potential resistance biomarker for anti-EGFR therapy, and warrants investigation as a molecular target.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4104657PMC
June 2014

Pan-erbB inhibition potentiates BRAF inhibitors for melanoma treatment.

Melanoma Res 2014 Jun;24(3):207-18

aDepartment of Pharmacology and Chemical Biology, University of Pittsburgh Cancer Institute Departments of bPathology cMedicine, University of Pittsburgh dDepartment of Dermatology, University of Pittsburgh Medical Center eDepartment of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania fDepartment of Internal Medicine, Division of Epidemiology and Biostatistics, University of New Mexico, Albuquerque, New Mexico gDepartment of Pharmacology, University of Minnesota, Minneapolis, Minnesota, USA.

The BRAF inhibitor vemurafenib is currently used for treating patients with BRAF V600E mutant melanoma. However, the responses to vemurafenib are generally partial and of relatively short duration. Recent evidence suggests that activation of the epidermal growth factor receptor (EGFR)/erbB signaling pathway may be responsible for the development of BRAF inhibitor resistance in melanoma patients. In this study, we characterized the erbB family of receptors and ligands in melanoma cell lines and examined whether targeting both BRAF and erbB provided enhanced antitumor activity in BRAF mutant melanoma. Variable levels of erbB2, erbB3, and truncated erbB4 were expressed in both BRAF wildtype and mutant melanoma cells with no significant differences between wildtype and mutant lines. EGFR was rarely expressed. Neuregulin 3 and neuregulin 4 were the major erbB ligands released by melanoma cells. Multi-erbB targeting with the irreversible tyrosine kinase inhibitor canertinib exerted a more effective growth inhibitory effect in both BRAF wildtype and mutant melanoma cells compared with the single-erbB or dual-erbB targeting inhibitors, gefitinib, erlotinib, and lapatinib. Canertinib inhibited both EGF-induced and neuregulin 1-induced erbB downstream signaling in both mutant and wildtype cell lines. However, canertinib induced apoptosis and sub-G1 arrest only in mutant cells. Canertinib statistically increased the antiproliferative effects of vemurafenib in the BRAF mutant melanoma cell lines while little or no enhanced effect was observed with the combination treatment in the wildtype cell lines. A combined inhibition strategy targeting BRAF together with multiple erbB family kinases is potentially beneficial for treating BRAF V600E mutant melanoma. Wildtype BRAF melanoma may also benefit from a multi-erbB kinase inhibitor.
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http://dx.doi.org/10.1097/CMR.0000000000000060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394744PMC
June 2014