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Lung cancer chemoprevention, especially in high-risk former smokers, has great potential to reduce lung cancer incidence and mortality. Thiazolidinediones prevent lung cancer in preclinical studies, and diabetics receiving thiazolidinediones have lower lung cancer rates which led to our double-blind, randomized, phase II placebo-controlled trial of oral pioglitazone in high-risk current or former smokers with sputum cytologic atypia or known endobronchial dysplasia. Bronchoscopy was performed at study entry and after completing 6 months of treatment. Biopsies were histologically scored, and primary endpoint analysis tested worst biopsy scores (Max) between groups; Dysplasia index (DI) and average score (Avg) changes were secondary endpoints. Biopsies also received an inflammation score. The trial accrued 92 subjects (47 pioglitazone, 45 placebo), and 76 completed both bronchoscopies (39 pioglitazone, 37 placebo). Baseline dysplasia was significantly worse for current smokers, and 64% of subjects had mild or greater dysplasia at study entry. Subjects receiving pioglitazone did not exhibit improvement in bronchial dysplasia. Former smokers treated with pioglitazone exhibited a slight improvement in Max, while current smokers exhibited slight worsening. While statistically significant changes in Avg and DI were not observed in the treatment group, former smokers exhibited a slight decrease in both Avg and DI. Negligible Avg and DI changes occurred in current smokers. A trend toward decreased Ki-67 labeling index occurred in former smokers with baseline dysplasia receiving pioglitazone. While pioglitazone did not improve endobronchial histology in this high-risk cohort, specific lesions showed histologic improvement, and further study is needed to better characterize responsive dysplasia.

Tyrosine kinase inhibitors (TKIs) targeting EGFR-mutant lung cancers promote a range of tumor regression responses to yield variable residual disease, a likely incubator for acquired resistance. Herein, rapid transcriptional responses induced by TKIs early in treatment that associate with the range of patient responses were explored. RNAseq was performed on EGFR mutant cell lines treated in vitro with osimertinib and on tumor biopsies of eight EGFR mutant lung cancer patients before and after 2 weeks of TKI treatment. Data were evaluated for gene expression programs altered upon TKI treatment. Chemokine and cytokine expression were measured by ELISA and quantitative RT-PCR. IκB Kinase (IKK) and JAK-STAT pathway dependence was tested with pharmacologic and molecular inhibitors. Tumor sections were stained for the T-cell marker CD3. Osimertinib stimulated dynamic, yet wide-ranging interferon (IFN) program regulation in EGFR mutant cell lines. IL6 and CXCL10 induction varied markedly among the EGFR mutant cell lines and was sensitive to IKK and JAK-STAT inhibitors. Analysis of matched patient biopsy pairs revealed marked, yet varied enrichment of IFN transcriptional programs, effector immune cell signatures and T-cell content in treated tumors that positively correlated with time to progression in the patients. EGFR-specific TKIs induce wide-ranging IFN response program activation originating within the cancer cell. The strong association of IFN program induction and duration of clinical response indicates that the TKI-induced IFN program instructs variable recruitment and participation of immune cells in the overall therapeutic response.

Introduction Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use. Methods The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature’s ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature. Results Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p Conclusions These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma.

Background Dabrafenib and trametinib combination therapy is approved for the treatment of patients with BRAF V600E positive tumors including melanoma and lung cancer. The effect of BRAF and MEK inhibitors on the immune system is not fully understood although a number of case reports indicate autoimmune side effects related to the use of these drugs. Here, we discuss a case of a patient diagnosed with granulomatosis with polyangiitis (GPA) shortly after starting treatment with dabrafenib and trametinib for BRAF V600E positive metastatic lung adenocarcinoma. Case presentation A 57 years old female patient was diagnosed with recurrent lung adenocarcinoma following initial lobectomy for early stage disease. A BRAF V600E mutation was identified at the time of recurrence and she received combination dabrafenib and trametinib therapy. Shortly after commencement of treatment, she developed persistent fevers necessitating withholding both drugs. Pyrexia continued and was followed by left vision loss and acute kidney injury. Further rheumatological workup led to the unifying diagnosis of GPA. The patient was then treated with rituximab for GPA to the present date while all antineoplastic drugs were held. Lung cancer oligoprogression was addressed with radiation therapy and has not required further systemic treatment whereas GPA has been controlled to-date with rituximab. Conclusions This case report raises awareness among clinicians treating patients with lung cancer for the possibility of triggering a flare of autoimmune diseases like GPA in patients with BRAF V600E positive lung cancer receiving treatment with BRAF directed therapy.

Squamous cell lung cancer arises from premalignant dysplasias characterized by squamous replacement of the normal mucociliary airway epithelium. Individuals with persistent, compared to regressive, dysplasias are at increased risk for lung cancer. Most studies have focused on genomic changes accrued in dysplasia, but the failure of normal epithelial repair has not been addressed. We hypothesized that basal progenitor cells, responsible for normal repair, are impaired in dysplasia. Functional analysis of basal progenitors from dysplastic bronchial biopsies showed decreased self-renewal and multipotentiality compared to non-dysplastic biopsies. The stable prostacyclin analog iloprost, the only drug to have improved dysplasia in a clinical trial, also rescued progenitor function and reversed dysplasia in a subset of lesions. In a series of 32 dysplastic lesions followed in 19 subjects who underwent serial bronchoscopic biopsies, baseline progenitor function predicted dysplasia persistence or regression. Epithelial repair dysfunction is an early and potentially manipulable event in bronchial premalignancy.

Analysis of a large group of patients with multifocal premalignant disease by Krysan and colleagues in this issue of Cancer Research provides an informative view of the processes that may underlie progression of these lesions to invasive adenocarcinoma of the lung. The identification of the type and distribution of mutational changes reveals that common processes may be occurring within individuals but that these are generally unique between patients at risk for developing lung cancer. Furthermore, predicted neoantigens are identified and associated with characteristics of immune infiltrates supporting the role of alterations in adaptive immune surveillance in progression of these premalignant lesions. These findings provide critical insights that will help establish a foundation of knowledge for developing personalized prevention strategies with the potential to significantly impact overall mortality in lung cancer.See related article by Krysan et al., p. 5022

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.

Lung cancer is the leading cause of cancer death worldwide and global burden could be reduced through targeted application of chemoprevention. The development of squamous lung carcinoma has been linked with persistent, high-grade bronchial dysplasia. Bronchial histology improved in former smokers in a chemoprevention trial with the prostacyclin analogue iloprost. Prostacyclin acts through peroxisome proliferator-activated receptor gamma (PPARγ) to reverse epithelial to mesenchymal transition and promote anticancer signaling. We hypothesized that the prostacyclin signaling pathway and EMT could provide response markers for prostacyclin chemoprevention of lung cancer. Human bronchial epithelial cells were treated with cigarette smoke condensate (CSC) or iloprost for 2 weeks, CSC for 16 weeks, or CSC for 4 weeks followed by 4 weeks of CSC and/or iloprost, and RNA was extracted. Wild-type or prostacyclin synthase transgenic mice were exposed to 1 week of cigarette smoke or one injection of urethane, and RNA was extracted from the lungs. We measured potential markers of prostacyclin and iloprost efficacy in these models. We identified a panel of markers altered by tobacco carcinogens and inversely affected by prostacyclin, including PPARγ, 15PGDH, CES1, COX-2, ECADHERIN, SNAIL, VIMENTIN, CRB3, MIR34c, and MIR221. These data introduce a panel of potential markers for monitoring interception of bronchial dysplasia progression during chemoprevention with prostacyclin. Chemoprevention is a promising approach to reduce lung cancer mortality in a high-risk population. Identifying markers for targeted use is critical for success in future clinical trials of prostacyclin for lung cancer chemoprevention. Cancer Prev Res; 11(10); 643–54. ©2018 AACR.

Persistent bronchial dysplasia is associated with increased risk of developing invasive squamous cell carcinoma (SCC) of the lung. In this study, we hypothesized that differences in gene expression profiles between persistent and regressive bronchial dysplasia would identify cellular processes that underlie progression to SCC. RNA expression arrays comparing baseline biopsies from 32 bronchial sites that persisted/progressed to 31 regressive sites showed 395 differentially expressed genes [ANOVA, FDR ≤ 0.05). Thirty-one pathways showed significantly altered activity between the two groups, many of which were associated with cell-cycle control and proliferation, inflammation, or epithelial differentiation/cell–cell adhesion. Cultured persistent bronchial dysplasia cells exhibited increased expression of Polo-like kinase 1 (PLK1), which was associated with multiple cell-cycle pathways. Treatment with PLK1 inhibitor induced apoptosis and G2–M arrest and decreased proliferation compared with untreated cells; these effects were not seen in normal or regressive bronchial dysplasia cultures. Inflammatory pathway activity was decreased in persistent bronchial dysplasia, and the presence of an inflammatory infiltrate was more common in regressive bronchial dysplasia. Regressive bronchial dysplasia was also associated with trends toward overall increases in macrophages and T lymphocytes and altered polarization of these inflammatory cell subsets. Increased desmoglein 3 and plakoglobin expression was associated with higher grade and persistence of bronchial dysplasia. These results identify alterations in the persistent subset of bronchial dysplasia that are associated with high risk for progression to invasive SCC. These alterations may serve as strong markers of risk and as effective targets for lung cancer prevention. Significance: Gene expression profiling of high-risk persistent bronchial dysplasia reveals changes in cell-cycle control, inflammatory activity, and epithelial differentiation/cell–cell adhesion that may underlie progression to invasive SCC. Cancer Res; 78(17); 4971–83. ©2018 AACR.

With more than 150,000 deaths per year in the US alone, lung cancer has the highest number of deaths for any cancer. These poor outcomes reflect a lack of treatment for the most common form of lung cancer, non-small cell lung carcinoma (NSCLC). Lung adenocarcinoma (ADC) is the most prevalent subtype of NSCLC, with the main oncogenic drivers being KRAS and epidermal growth factor receptor (EGFR). Whereas EGFR blockade has led to some success in lung ADC, effective KRAS inhibition is lacking. KRAS-mutant ADCs are characterized by high levels of gel-forming mucin expression, with the highest mucin levels corresponding to worse prognoses. Despite these well-recognized associations, little is known about roles for individual gel-forming mucins in ADC development causatively. We hypothesized that MUC5AC/Muc5ac, a mucin gene known to be commonly expressed in NSCLC, is crucial in KRAS/Kras-driven lung ADC. We found that MUC5AC was a significant determinant of poor prognosis, especially in patients with KRAS-mutant tumors. In addition, by using mice with lung ADC induced chemically with urethane or transgenically by mutant-Kras expression, we observed significantly reduced tumor development in animals lacking Muc5ac compared with controls. Collectively, these results provide strong support for MUC5AC as a potential therapeutic target for lung ADC, a disease with few effective treatments.

IntroductionEarly detection of lung cancer in high-risk individuals reduces mortality. Low-dose spiral computed tomography (LDCT) is the current standard but suffers from an exceedingly high false-positive rate (>96%) leading to unnecessary and potentially dangerous procedures. We, therefore, set out to develop a simple, noninvasive, and quantitative assay to detect lung cancer.MethodsThis proof-of-concept study evaluated the sensitivity/specificity of the CyPath Early Lung Cancer Detection Assay to correctly classify LDCT-confirmed cohorts of high-risk control (n = 102) and cancer (n = 26) subjects. Fluorescence intensity parameters of red fluorescent cells (RFCs) from tetra (4-carboxyphenyl) porphyrin (TCPP)-labeled lung sputum samples and subjects’ baseline characteristics were assessed for their predictive power by multivariable logistic regression. A receiver operating characteristic curve was constructed to evaluate the sensitivity/specificity of the CyPath assay.ResultsRFCs were detectable in cancer subjects more often than in high-risk ones (p = 0.015), and their characteristics differed between cohorts. Two independent predictors of cancer were the mean of RFC average fluorescence intensity/area per subject (p < 0.001) and years smoked (p = 0.003). The CyPath-based classifier had an overall accuracy of 81% in the test population; false-positive rate of 40% and negative predictive value of 83%.ConclusionsThe tetra (4-carboxyphenyl) porphyrin -based CyPath assay correctly classified study participants into cancer or high-risk cohorts with considerable accuracy. Optimizing sputum collection, sample reading, and refining the classifier should improve sensitivity and specificity. The CyPath assay thus has the potential to complement LDCT screening or serve as a stand-alone approach for early lung cancer detection.

Lung adenocarcinoma (AdC) and lung squamous cell carcinoma (SCC) are the most common non-small cell lung cancer (NSCLC) subtypes, however, most genetic mouse models of lung cancer produce predominantly, if not exclusively, AdC. Whether this is secondary to targeting mutations to the distal airway cells or to the use of activating Kras mutations that drive AdC formation is unknown. We previously showed that targeting Kras(G12D) activation and transforming growth factor β receptor type II (TGFβRII) deletion to airway basal cells via a keratin promoter induced formation of both lung AdC and SCC. In this study we assessed if targeting phosphatase and tensin homologue (PTEN) deletion to airway basal cells could initiate lung tumor formation or increase lung SCC formation. We found that PTEN deletion is capable of initiating both lung AdC and SCC formation when targeted to basal cells and although PTEN deletion is a weaker tumor initiator than Kras(G12D) with low tumor multiplicity and long latency, tumors initiated by PTEN deletion were larger and displayed more malignant conversion than Kras(G12D) initiated tumors. That PTEN deletion did not increase lung SCC formation compared to Kras(G12D) activation, suggests that the initiating genetic event does not dictate tumor histology when genetic alterations are targeted to a specific cell. These studies also confirm that basal cells of the conducting airway are capable of giving rise to multiple NSCLC tumor types.

Mesenchymal-epithelial transition factor gene (MET) gene copy number gain may be a predictive biomarker for mesenchymal-epithelial transition factor (MET) inhibition in lung cancer, but the most appropriate method and criteria for defining MET positivity are uncertain.MET copy number was assessed by fluorescence in situ hybridization in lung adenocarcinoma. Positivity criteria included mean MET per cell values greater than 5 (low [≥5 to6], intermediate [≥6 to7], and high [≥7]) and mean MET-to-chromosome 7 centromere ratios (MET/CEP7) of at least 1.8 (low [≥1.8 to ≤2.2], intermediate [2.2 to5], and high [≥5]). Associated clinical and molecular characteristics were captured.Of 686 cases, 99 (14%) had a mean MET per cell value of 5 or greater, 52 of 1164 (4.5%) had a MET/CEP7 ratio of 1.8 or higher. Other oncogenic drivers (in EGFR, KRAS, anaplastic lymphoma receptor tyrosine kinase gene [ALK], erb-b2 receptor tyrosine kinase 2 gene [ERBB2], BRAF, NRAS, ROS1, or ret proto-oncogene [RET]) were detectable in 56% of the group with a mean MET per cell value of 5 or higher and 47% of the group with a MET/CEP7 ratio of 1.8 or higher, suggesting that many MET-positive cases are not truly MET addicted. The rates of concomitant drivers in the groups of patients in the low, indeterminate, and high categories of mean MET per cell were 32 of 52 (62%), 12 of 19 (63%), and 11 of 27 (41%) (p = 0.2), and the rates of concomitant drivers in the low, intermediate, and high categories of MET/CEP7 ratios were 15 of 29 (52%), 9 of 18 (50%), and 0 of 4 (0%), respectively (p = 0.04). A MET/CEP7 ratio of 1.8 or higher in the absence of other oncogenes was associated with a higher rate of adrenal metastases (p = 0.03) but not with never-smoking status.A fluorescence in situ hybridization MET/CEP7 ratio of 5 or higher defined a "MET-positive" group with no oncogenic overlap. As this method and criteria are also associated with the highest response rate to MET inhibition, it represents the clearest definition of a MET copy number gain-addicted state. However, a MET-associated phenotype may also exist across cases with a MET/CEP7 of 1.8 or higher when no other oncogene overlap occurs.

Purpose: Lung adenocarcinoma and lung squamous cell carcinoma (SCC) are the most common non–small cell lung cancer (NSCLC) subtypes. This study was designed to determine whether reduced expression of TGFβ type II receptor (TGFβRII) promotes lung adenocarcinoma and SCC carcinogenesis. Experimental Design: We examined TGFβRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFβRII expression and clinicopathologic parameters. To determine whether sporadic TGFβRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFβRII deletion alone and in combination with oncogenic KrasG12D to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. Results: Reduced TGFβRII expression in human NSCLC is associated with male gender, smoking, SCC histology, reduced differentiation, increased tumor stage, increased nodal metastasis, and reduced survival. Homozygous or heterozygous TGFβRII deletion in mouse airway epithelia increases the size and number of KrasG12D-initiated adenocarcinoma and SCC. TGFβRII deletion increases proliferation, local inflammation, and TGFβ ligand elaboration; TGFβRII knockdown in airway epithelial cells increases migration and invasion. Conclusions: Reduced TGFβRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFβ1 expression. TGFβRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFβRII loss plays a causal role in lung carcinogenesis. That TGFβRII shows haploid insufficiency suggests that a 50% TGFβRII protein reduction would negatively impact lung cancer prognosis. Clin Cancer Res; 18(8); 2173–83. ©2012 AACR.

There are no established chemopreventive agents for lung cancer, the leading cause of cancer death in the United States. Prostacyclin levels are low in lung cancer and supplementation prevents lung cancer in preclinical models. We carried out a multicenter double-blind, randomized, phase II placebo-controlled trial of oral iloprost in current or former smokers with sputum cytologic atypia or endobronchial dysplasia. Bronchoscopy was performed at study entry and after completion of six months of therapy. Within each subject, the results were calculated by using the average score of all biopsies (Avg), the worst biopsy score (Max), and the dysplasia index (DI). Change in Avg was the primary end point, evaluated in all subjects, as well as in current and former smokers. The accrual goal of 152 subjects was reached and 125 completed both bronchoscopies (60/75 iloprost, 65/77 placebo). Treatment groups were well matched for age, tobacco exposure, and baseline histology. Baseline histology was significantly worse for current smokers (Avg 3.0) than former smokers (Avg 2.1). When compared with placebo, former smokers receiving oral iloprost exhibited a significantly greater improvement in Avg (0.41 units better, P = 0.010), in Max (1.10 units better, P = 0.002), and in DI (12.45%, P = 0.006). No histologic improvement occurred in current smokers. Oral iloprost significantly improves endobronchial histology in former smokers and deserves further study to determine if it can prevent the development of lung cancer. Cancer Prev Res; 4(6); 793–802. ©2011 AACR.

PurposeIdentification of new therapies in small cell lung cancer (SCLC) is urgently needed. Insulin-like growth factor 1 receptor (IGF1R) is a tyrosine kinase receptor implicated in the pathogenesis of several malignancies and is potentially an attractive target for anticancer treatment. Knowledge about IGF1R protein expression, gene copy number, and the prognostic relevance of these features in SCLC is limited.MethodsWe analyzed IGF1R protein expression and gene copy number in primary tumors from 90 patients with SCLC (67 men and 23 women) who underwent pulmonary resection. IGF1R expression assessed by immunohistochemistry with H scores from 0 to 400 was evaluable in 84 patients and IGF1R gene copy number assessed by silver in situ hybridization technique in 81 patients.ResultsMedian H score for IGF1R protein expression was 88 (range, 0–400), and the proportion of positive immunostaining using cutoff H score of 10 was 74%. Increased IGF1R gene copy number (an average of four or more copies per cell) was found in 15 cases (18.5%), five of whom (6.2%) showed gene amplification. There was a significant correlation between protein expression and gene copy number (r = 0.49, p < 0.005). IGF1R expression and gene copy number did not associate with clinicopathological factors such as patient age, tumor size, lymph node involvement, stage, and survival.ConclusionsSCLC is characterized by frequent high-IGF1R protein expression, increased gene copy number, and occasional occurrence of true gene amplification. These features may have important implications for future anti-IGF1R therapeutic approaches.

Lung cancer remains the leading cause of cancer death in the United States, and chemoprevention offers an appealing area of investigation in the face of limited therapeutic success. Improvement in endobronchial histology was demonstrated in former smokers after oral iloprost chemoprevention in a phase II clinical trial. Of the 48 patients who received iloprost in the chemoprevention trial, 23 had regressive histology and 25 had stable or progressive histology. Identifying markers that predict which patients will respond to treatment will help refine target populations for future trials and clinical applications. Markers of drug activity will also facilitate clinical application by allowing monitoring during treatment. Preclinical studies indicate that iloprost, a prostacyclin analogue, acts through peroxisome proliferator activated receptor gamma (PPAR) to reduce transformed lung epithelial cell growth and markers of epithelial-to-mesenchymal transition (EMT). We hypothesize that PPAR expression status predicts response to prostacyclin chemoprevention and that expression changes in known downstream targets of PPAR and prostacyclin indicate response to prostacyclin treatment. Prostacyclin may also induce expression of PPAR, leading to increased antitumor signaling. We found that expression of PPAR and markers of prostacyclin signaling (15pgdh, CES1, and PTGS2) and EMT markers (Snail, Vimentin, Ecadherin, and CRB3) changed in cultured human bronchial epithelial cells (HBEC) in response to cigarette smoke condensate (CSC) and that iloprost reversed these changes. Expression in mice also changed in response to cigarette smoke carcinogens and prostacyclin. miR-34c is inversely correlated with bronchial histology grade and suppresses lung cancer progression. In our study, the level of miR-34c was lower in the NSCLC cell line H322 compared to HBEC. In H322, miR-34c increased with PPAR agonist treatment and decreased with PPAR antagonist treatment. In HBEC and mouse models of lung cancer chemoprevention, miR-34c increased with prostacyclin and decreased with cigarette smoke carcinogen exposure. In cultured primary progressive dysplastic bronchial epithelial cells, treatment with iloprost resulted in similar expression changes in Vimentin, Snail, Ecadherin, CRB3, CES1, and miR-34c. This study has the potential to improve iloprost lung cancer chemoprevention by allowing future trials to more effectively target high-risk patients, by providing a clinical biomarker for identification of chemoprevention candidates, and by identifying biomarkers for intermediate monitoring of response. Future studies will test expression of these markers in lung tissue biopsy and liquid biopsy samples from the oral and inhaled iloprost chemoprevention trials. Citation Format: Melissa New, Debbie McArthur, Lori Nield, Dan Merrick, Robert L. Keith, Meredith A. Tennis. Biomarkers for precision application of prostacyclin lung cancer chemoprevention [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B35.

No clinically effective chemoprevention for lung cancer has been found. Angiogenesis is an early feature of both adenocarcinoma and squamous cell lung cancer. We investigated the effects of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) inhibition on lung carcinogenesis in a murine model of adenocarcinoma. The VEGFR-2 tyrosine kinase inhibitor, vandetanib, was given to FVB/N mice in chow for 7 days at varying doses to show pharmacologic activity by inhibition of VEGF-mediated VEFGR-2 and ERK phosphorylation. Plasma levels corroborated adequate dosage. For chemoprevention experiments, mice were injected i.p. with 1 mg/g of urethane, a carcinogen found in tobacco smoke. Chow containing vandetanib, 75 mg/kg/d, or control chow was given to mice, starting 7 days after urethane administration. Sixteen weeks after urethane injection, mice were sacrificed, tumors enumerated and measured. Vandetanib resulted in reductions in tumor multiplicity (6.5 ± 0.86 versus 1.0 ± 0.30, P = 0.001) and average tumor volume (0.85 ± 0.10 versus 0.15 ± 0.09 mm3, P = 0.001), but not incidence (71% versus 100%, P = ns), compared with control. As vandetanib has other activities besides VEGFR-2 tyrosine kinase inhibition, we gave the anti–VEGFR-2 monoclonal antibody, DC101, for weeks 11 to 15 of a urethane carcinogenesis protocol with an arrest in tumor volume increase, but no change in multiplicity or incidence. Further investigation of the chemopreventive effect of vandetanib and other VEGF signaling inhibitors is needed. Cancer Prev Res; 3(9); 1141–7. ©2010 AACR.

Background: There is a need for early detection methods for lung cancer. Radiologic imaging may be more sensitive for peripheral cancers than for cancers arising in the central airways, from which bronchial epithelial cells are exfoliated into the sputum. Methods: Sputum samples were collected at baseline and periodically thereafter in a cohort of smokers and former smokers with chronic obstructive lung disease. The association between cytologic atypia and incident lung cancer was assessed by hazard ratios (HR; 95% confidence intervals) using Cox regression and by odds ratios (95% confidence intervals) using logistic regression, adjusting for potential confounding factors. Results: We observed 174 incident lung cancers in a cohort of 2,521 people over 9,869 person-years of observation. Risk for incident lung cancer was increased among those with cytologic atypia graded as moderate or worse (adjusted HR, 2.37; 1.68-3.34). The association between sputum atypia and lung cancer incidence was greatest for those sputum samples collected 5 months or less before the diagnosis of lung cancer (odds ratio, 10.32; 5.34-19.97). The association was substantially stronger for squamous cell lung cancers (HR, 5.13; 2.89-9.10) than for adenocarcinomas (HR, 1.85; 0.94-3.65). Conclusion: Cytologic atypia is a marker for increased lung cancer risk. These cytologic changes seem to arise from late events that are most apparent for cancers arising in the central respiratory airways. Whether cytologic atypia might complement radiologic imaging in a combined approach to lung cancer, early detection requires additional evaluation of those two methods used together. (Cancer Epidemiol Biomarkers Prev 2008;17(1):158–63)

Purpose: Lung cancer is preceded by a premalignant phase during which intervention could decrease associated morbidity and mortality. Molecular characterization of factors involved in controlling progression of bronchial dysplasias will provide markers of premalignant change and identify targets for chemoprevention. Experimental Design: Immunohistochemical analysis of epidermal growth factor receptor (EGFR; c-ErbB1/EGFR), HER-2/neu (c-ErbB2/HER-2), Ki-67, and minichromosome maintenance protein 2 (MCM2) expression in bronchial dysplasia was undertaken to characterize molecular alterations associated with the progression of these lesions in 268 bronchoscopically obtained biopsies from 134 subjects. Results: Analysis of biopsies with the most severe diagnosis from each subject showed a linear relationship between increasing marker expression and severity of dysplastic change for EGFR (P < 0.001), Ki-67 (P < 0.001), and MCM2 (P = 0.001) but not HER-2 (P = 0.102). Increased expression of either EGFR or HER-2 was associated with increased levels of Ki-67 and MCM2 expression, and combined overexpression of these receptors was associated with the highest levels of proliferation, suggesting a synergistic effect. Finally, the lack of an associated trend toward increased EGFR expression when comparing the worst and best biopsies within each subject indicated a potential field effect in the induction of EGFR expression. Conclusions: The results suggest a prominent role for EGFR overexpression in the development and progression of bronchial dysplasia and provide rationale for exploring inhibition of EGFR signaling in lung cancer chemoprevention.

Neoangiogenesis is required for the growth of invasive lung carcinoma, however, the role of angiogenesis in the progression of premalignant changes to carcinoma of the lung is less clear. We have evaluated vascular endothelial growth factor (VEGF) expression and microvessel densities (MVDs) in 62 bronchoscopic biopsies from normal, reactive (basal cell hyperplasia (BCH)) and dysplastic bronchial epithelium and in tissue from twenty-seven invasive lung carcinomas in an effort to demonstrate angiogenic activity in these preneoplastic lesions and determine whether it is associated with increased bronchial epithelial VEGF expression. MVDs and VEGF RNA expression measured by quantitative RT-PCR were found to be elevated in comparison to normal bronchial tissue in bronchial dysplasias and invasive lung carcinomas but not in basal cell hyperplasias. Immunohistochemical (IHC) analyses revealed that expression of VEGF arose predominantly from bronchial epithelium. ELISA analysis of lung tumor tissue showed that elevated VEGF protein expression correlated with VEGF RNA levels (r=0.59, p=0.004). Increased expression of VEGF RNA was also found in histologically normal bronchial mucosa from patients with either dysplasia at other sites or a history of heavy tobacco use suggesting a possible field effect in regard to the elaboration of VEGF. Furthermore, analysis of VEGF isoforms and VEGF receptors by semi-quantitative RT-PCR in dysplastic and invasive lesions revealed characteristic altered patterns of expression in dysplasia and early cancer as compared to normal tissue. These results indicate that angiogenesis develops early in lung carcinogenesis and is associated with overexpression of VEGF.

TPS9100 Background: Significant advances in the clinical outcomes of lung cancer patients have been achieved in part due to the identification of targetable driver mutations. The success of targeting oncogenic drivers with TKIs has allowed for improvements in response rates, progression free survival and overall survival. Despite these improvements, patients ultimately relapse. Acquired resistance has been evaluated in post-progression tumor samples and is caused by a variety of mechanisms including secondary resistance mutations, gene copy number gains, gene amplification, or bypass pathway activation. Residual tumor burden with surviving cancer cells are the origin of the ultimate tumor progression. Studies analyzing signaling pathways and other markers of these tumor cells or microenvironmental cells in tumor samples before clinical resistance develops are needed to determine how best to target this compartment. Methods: Eligible patients will have a new diagnosis of advanced NSCLC with an EGFR TKI sensitizing mutation. Additional inclusion criteria include ECOG 0-2, a tumor measurable by RECIST 1.1 and at least 2cm in one dimension, and tumor site that is accessible and safe to biopsy. Patients will undergo a pretreatment target lesion biopsy, begin treatment with an EGFR TKI then receive a second biopsy of the same target lesion after two weeks of therapy. Our primary objective is to identify differences between pretreatment and early treatment tumor samples. To do this we will be using a combination of RNA seq, multiplex protein expression analysis and proximity ligation assays. Secondary objectives include identification of predictive markers that can be used to determine which tumors will undergo epithelial to mesenchymal transition or activate other survival pathways and determination of the success rate and adverse event rate of repeat biopsy. Patients are currently being enrolled at a single academic institution however, additional clinical site openings are pending. Our goal enrollment for this study is 47 patients to achieve 20 paired biopsy samples. We will present updated enrollment and demographic information. Clinical trial information: NCT03042221.

Chronic inflammation is a risk factor for lung cancer, and low-dose aspirin intake reduces lung cancer risk. However, the roles that specific inflammatory cells and their products play in lung carcinogenesis have yet to be fully elucidated. In mice, alveolar macrophage numbers increase as lung tumors progress, and pulmonary macrophage programing changes within 2 weeks of carcinogen exposure. To examine how macrophages specifically affect lung tumor progression, they were depleted in mice bearing urethane-induced lung tumors using clodronate-encapsulated liposomes. Alveolar macrophage populations decreased to ≤50% of control levels after 4–6 weeks of liposomal clodronate treatment. Tumor burden decreased by 50% compared to vehicle treated mice, and tumor cell proliferation, as measured by Ki67 staining, was also attenuated. Pulmonary fluid levels of insulin-like growth factor-I, CXCL1, IL-6, and CCL2 diminished with clodronate liposome treatment. Tumor-associated macrophages expressed markers of both M1 and M2 programing in vehicle and clodronate liposome-treated mice. Mice lacking CCR2 (the receptor for macrophage chemotactic factor CCL2) had comparable numbers of alveolar macrophages and showed no difference in tumor growth rates when compared to similarly treated wild-type mice suggesting that while CCL2 may recruit macrophages to lung tumor microenvironments, redundant pathways can compensate when CCL2/CCR2 signaling is inactivated. Depletion of pulmonary macrophages rather than inhibition of their recruitment may be an advantageous strategy for attenuating lung cancer progression.

Non-small-cell lung cancer (NSCLC) is a common malignancy with a poor prognosis. Despite progress targeting oncogenic drivers, there are no therapies targeting tumor-suppressor loss. Smad4 is an established tumor suppressor in pancreatic and colon cancer; however, the consequences of Smad4 loss in lung cancer are largely unknown. We evaluated Smad4 expression in human NSCLC samples and examined Smad4 alterations in large NSCLC data sets and found that reduced Smad4 expression is common in human NSCLC and occurs through a variety of mechanisms, including mutation, homozygous deletion and heterozygous loss. We modeled Smad4 loss in lung cancer by deleting Smad4 in airway epithelial cells and found that Smad4 deletion both initiates and promotes lung tumor development. Interestingly, both Smad4(-/-) mouse tumors and human NSCLC samples with reduced Smad4 expression demonstrated increased DNA damage, whereas Smad4 knockdown in lung cancer cells reduced DNA repair and increased apoptosis after DNA damage. In addition, Smad4-deficient NSCLC cells demonstrated increased sensitivity to both chemotherapeutics that inhibit DNA topoisomerase and drugs that block double-strand DNA break repair by non-homologous end joining. In sum, these studies establish Smad4 as a lung tumor suppressor and suggest that the defective DNA repair phenotype of Smad4-deficient tumors can be exploited by specific therapeutic strategies.

Lung cancers express lower levels of prostacyclin than normal lung tissues. Prostacyclin prevents lung cancer in a variety of mouse models. A randomized phase II trial comparing oral iloprost (a prostacyclin analog) with placebo in high-risk subjects showed improvement in bronchial histology in former, but not current, smokers. This placebo-controlled study offered the opportunity for investigation of other potential intermediate endpoint and predictive biomarkers to incorporate into chemoprevention trials. Matched bronchial biopsies were obtained at baseline and at 6-month follow-up from 125 high-risk individuals who completed the trial: 31/29 and 37/28 current/former smokers in the iloprost and placebo arm, respectively. We analyzed the expression of 14 selected miRNAs by Real Time PCR in 496 biopsies. The expression of seven miRNAs was significantly correlated with histology at baseline. The expression of miR-34c was inversely correlated with histology at baseline (P < 0.0001) and with change in histology at follow-up (P = 0.0003), independent of treatment or smoking status. Several miRNAs were also found to be differentially expressed in current smokers as compared with former smokers. In current smokers, miR-375 was upregulated at baseline (P < 0.0001) and downregulated after treatment with iloprost (P = 0.0023). No miRNA at baseline reliably predicted a response to iloprost. No biomarker predictive of response to iloprost was found. MiR-34c was inversely correlated with baseline histology and with histology changes. Mir-34c changes at follow-up could be used as a quantitative biomarker that parallels histologic response in formalin-fixed bronchial biopsies in future lung cancer chemoprevention studies. Cancer Prev Res; 6(2); 100–8. ©2012 AACR.

9065Background: Non-small cell lung cancer (NSCLC) patients (pts) harboring ROS1 (ROS1+)and ALK gene fusions (ALK+) treated with oncogene-directed therapies experience significant improvements in r...

Patients with chronic obstructive pulmonary disease (COPD) are at an increased risk for the development of lung cancer, the mechanisms for which are incompletely understood. We hypothesized that the hypoxic pulmonary microenvironment present in COPD would augment lung carcinogenesis. Mice were subjected to chemical carcinogenesis protocols and placed in either hypoxia or normoxia. Mice exposed to chronic hypoxia developed tumors with increased volume compared with normoxic controls. Both lungs and tumors from hypoxic mice showed a preferential stabilization of HIF-2α and increased expression of VEGF-A, FGF2, and their receptors as well as other survival, proliferation, and angiogenic signaling pathways regulated by HIF-2α. We showed that tumors arising in hypoxic animals have increased sensitivity to VEGFR-2/EGFR inhibition, as chemoprevention with vandetanib showed markedly increased activity in hypoxic mice. These studies showed that lung tumors arising in a hypoxic microenvironment express increased growth, angiogenic, and survival signaling that could contribute to the increased lung cancer risk in COPD. Furthermore, the differential sensitivity of tumors arising in hypoxia to VEGFR-2/EGFR inhibition suggests that the altered signaling present in tumors arising in hypoxic lung might be therapeutically exploited in patients with underlying COPD. Cancer Prev Res; 5(8); 1061–71. ©2012 AACR.

Tumor-associated macrophages (TAMs) encourage and coordinate neoplastic growth. In late stage human lung adenocarcinoma, TAMs exhibited mixed M1 (classical; argI(low)iNOS(high)) and M2 (alternative; argI(high)iNOS(low)) polarization based on arginine metabolism. In several murine cancer models including chemically and genetically-induced primary lung tumors, prostate tumors, colon xenografts, and lung metastases, TAMs expressed argI(high)iNOS(low) early during tumor formation; argI(low)iNOS(high) polarization also occurred during malignancy in some models. In a chemically-induced lung tumor model, macrophages expressed argI(high)iNOS(low) within one week after carcinogen treatment, followed by similar polarization of bone marrow-derived monocytes (BDMCs) a few days later. TAMs surrounding murine prostate tumors also expressed argI(high)iNOS(low) early during tumorigenesis, indicating that this polarization is not unique to neoplastic lungs. In a human colon cancer xenograft model, the primary tumor was surrounded by argI(high)iNOS(low)-expressing TAMs, and BDMCs also expressed argI(high)iNOS(low), but pulmonary macrophages adopted argI(high)iNOS(low) polarization only after tumors metastasized to the lungs. Persistence of tumors is required to maintain TAM polarization. Indeed, in both conditional mutant Kras- and FGF10-driven models of lung cancer, mice expressing the transgene develop lung tumors that regress rapidly when the transgene is silenced. Furthermore, pulmonary macrophages expressed argI(high)iNOS(low) on tumor induction, but then returned to argI(low) iNOS(low) (no polarization) after tumors regressed. Manipulating TAM function or depleting TAMs may provide novel therapeutic strategies for preventing and treating many types of cancer.

Squamous cell lung cancer (SCC) is the second leading cause of lung cancer death in the US with a 5-year survival rate of 16.8%. Since most SCC is diagnosed at a late stage, chemoprevention or early detection could improve survival. SCC develops through a series of histological alterations including dysplasia and carcinoma in situ. Previous studies have identified genetic changes in epithelial progenitor cells in dysplastic lesions. The goal of this project is to establish a correlation between specific genetic changes in epithelial progenitors and their function. A tissue stem cell is a progenitor cell that is capable of long-term self-renewal as well as differentiation to all the cell-types that make up a specific tissue and therefore is multipotential. Because the normal function of a progenitor cell is to repair and regenerate a native epithelium after injury, histologic changes in dysplasia led us hypothesize that “normal function of airway progenitors is altered in dysplasia”. To address this hypothesis we purified and established clonal cultures of basal progenitors from normal, moderate and severely dysplastic biopsies and compared their self-renewal and differentiation abilities. These analyses revealed that the self-renewal capacity of progenitor cells decreased as dysplasia grade increased. The progenitors from normal biopsies differentiated to all three cell-types (basal, secretory or mucin-secreting and ciliated) of the airways. In contrast, progenitors from dysplasia generated increased numbers of basal cells (p Citation Format: Moumit Ghosh, Ichiro Nakachi, Robert L. Keith, Daniel T. Merrick, Wilbur A. Franklin, York E. Miller. Malfunction and mutation of airway stem/progenitor cells in preneoplastic bronchial dysplasia. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B19.

Purpose of review Given the poor prognosis associated with lung cancer, the ability to diagnose lung cancer in its early stages is considered crucial to achieving decreased lung cancer mortality. Herein, we discuss recent advances in biomarker discovery and evaluation of their potential application in the clinical setting. Recent findings Novel potential biomarkers have been identified via new techniques including cDNA microarray analysis, comparative genomic hybridization, and proteomics. These factors have been evaluated in various validation studies including tissue microarrays, RT-PCR, and assays of bioactivity. The characterization of these potential biomarkers through analysis of pathways that have been associated with neo-plastic transformation may help to identify the precursor lesions that are associated with subsequent progression to invasive carcinoma. In addition, the past year has also produced intriguing results regarding the detection of biomarkers in easily accessible screening specimens such as sputa, serum, and exhaled breath. Recent advances in these aspects of biomarker identification for the early detection of lung cancer are reviewed. Summary Identification of new candidate biomarkers and improved applications of previously detected biomarkers show great promise for the ultimate establishment of practical lung cancer screening. While recent studies engender optimism for the creation of clinically applicable screening tests, the biomarkers that have been identified need larger, follow-up validational studies and further characterization as to their biologic importance.

Transforming growth factor-β (TGF-β) is a pluripotent regulator of epithelial growth and homeostasis and functions as a tumor suppressor in numerous malignancies. TGF-β signaling defects are common in many malignancies, including non–small cell lung cancer (NSCLC); however, the mechanisms by which defective TGF-β signaling promotes lung cancer growth and development are relatively unknown. We generated a novel NSCLC mouse model by simultaneously activating oncogenic KrasG12D expression and deleting TGF-β receptor type II (TGF-βRII) in airway epithelial cells. Genetic manipulation was targeted to the airway by K5Cre*PR, which uses a keratin 5 (K5) promoter to direct expression of an RU486-inducible, Cre recombinase-progesterone receptor fusion protein to epithelial cells. K5Cre*PR.lox-stop-lox-KrasG12D.TGF-βRII flox/flox mice treated with tracheal RU486 develop multifocal NSCLC beginning at approximately 32 weeks of age. Interestingly, these animals develop both thyroid transcription factor (TTF)-positive adenocarcinomas and K5-positive squamous cell carcinomas. TGF-βRII deletion increases tumor multiplicity from 3.2 ± 1.7 to 49 ± 10 tumors per animal and increases average tumor size from 0.71 to 1.04 mm, compared with tumors generated in K5Cre*PR.LSL-KrasG12D mice (P < 0.05 for both comparisons). In addition, K5Cre*PR.LSL-KrasG12D.TGFβRII flox/flox mice have both increased serum and bronchoalveolar lavage (BAL) TGF-β1 levels as well as increased BAL macrophages. Mechanistically, TGF-βRII deletion increases the multiplicity and growth of K-ras–induced lung cancers through mechanisms involving increased proliferation and increased inflammation.

Lung cancer is the leading cause of cancer deaths in developed countries. The poor prognosis associated with this disease is closely related to the fact that most lung cancer patients are not identified until their malignancy has reached an advanced stage. Recent advances have added to the understanding of the morphological and molecular characteristics of preinvasive bronchial lesions and early lung cancers. Such information is being used to provide new tests for the detection of lung cancer at early or preinvasive stages, and for identifying targets for therapeutic intervention that can prevent progression to advanced disease. Laser induced fluorescence endoscope bronchoscopy has improved the sensitivity with which preinvasive dysplastic bronchial lesions and early invasive malignancies can be detected. Morphological features of such lesions have been described and can be monitored by follow-up bronchoscopies in order to validate potential chemoprevention treatments. Distinct morphological characteristics such as angiogenic squamous dysplasia also suggest that processes like angiogenesis are present early in the development of lung cancer. Furthermore, tissue obtained from these early lesions has been used to describe alterations in the expression of a number of factors that distinguish these early lesions from normal bronchial epithelium. This could provide molecular markers and targets for the detection and treatment of early lung cancer. Studies to detect these alterations by polymerase chain reaction and/or immunhistochemical analyses of easily obtained specimens such as sputa are helping identifing molecular markers that could be utilized in effective screening programmes. The current article reviews new findings regarding the molecular biology of preinvasive bronchial lesions and early lung cancers, and describes new developments regarding their application in the early detection and chemoprevention of lung cancer.

Squamous cell carcinoma (SQ) of the lung causes approximately 400,000 deaths per year worldwide, and no specifically targeted treatments yet exist. We used the SOMAscan proteomic platform (which measures 1129 proteins with a median limit of detection of 40 fM and 5% CV) in a broad-based analysis of serum and tissue samples to gain insight into the distinctive drivers of SQ malignant growth. We discovered unique SQ markers and pathways that show early expression of invasion and metastasis signals. Strong SQ signals were revealed. Our multi-center serum study of 94 non-small cell lung cancer (NSCLC) cases and 269 long-term smoker and benign pulmonary nodule controls resulted in the discovery of 60 lung cancer biomarkers and the development of a 7-marker diagnostic panel. This panel was validated in two independent cohorts. The AUC for detection of SQ carcinoma was 0.93 in training and 0.89 in the Univ. of Heidelberg validation set (56 SQ cases/27 benign nodule controls). Performance was confirmed with an AUC of 0.87 in an independent validation cohort assembled by the EDRN (25 SQ cases/20 benign nodules/52 smoker controls). This non-invasive test could be used as an adjunct to CT to detect rapidly growing SQ tumors that are disproportionately missed as interval cancers in CT screening studies. In addition, we analyzed 68 NSCLC tumor and matched non-tumor tissue lysates. This study consisted of 49 adenocarcinoma (AD) and 19 SQ tumors, 88% of which were Stage I or II. Proteomic comparisons of tumor/non-tumor or AD/SQ tissue samples performed using the Mann-Whitney test identified 79 tumor markers. Differences between tumor and non-tumor tissue were dominated by inflammatory, apoptotic and cell proliferation proteins. Just as we observed in the serum studies, the most common pattern was an increasing difference in protein levels from non tumor to AD to SQ. Of note, 24% of the proteins were only markers in SQ. These SQ-only markers are enriched for angiogenesis, cell proliferation and cell adhesion functions. Unexpected findings that the SQ proteome is dominated by proteins that promote malignancy and metastasis even in early stage tumors were revealed through broad proteomic profiling. These insights may lead to essential connections between the biochemical pathways altered in malignancy and tools for SQ cancer diagnosis and treatment. This abstract is also presented as Poster B14. Citation Format: Rachel Ostroff, Michael R. Mehan, Stephen Williams, Edward Brody, Harvey Pass, William Rom, Jill Siegfried, Thomas Muley, Wilbur Franklin, Dan Merrick, Adrie van Bokhoven, Holly Wolf, William Feser, Anna E. Barón, York Miller. Distinctive squamous cell carcinoma protein signatures. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr PR04.

Lung cancer remains the leading cause of cancer death in the United States and chemoprevention offers an appealing area of investigation in the face of limited therapeutic success. We recently demonstrated improvement in endobronchial histology in former smokers after oral iloprost treatment. Of the 48 patients who received iloprost in the chemoprevention trial, 23 had regressive histology and 25 had stable or progressive histology. Identifying markers that predict response to treatment will refine target populations for future trials and clinical applications. In vitro studies of NSCLC indicate that iloprost, a prostacyclin analogue, acts through the G-protein coupled receptor Frizzled 9 (Fzd9) instead of the prostacyclin receptor. We hypothesize that Fzd9 expression status predicts response to iloprost chemoprevention and that expression is modified by promoter hypermethylation. Patients who have lost Fzd9 expression will not respond to iloprost treatment. If Fzd9 is lost by methylation, a demethylating agent could restore Fzd9 expression and sensitivity to iloprost chemoprevention. In a urethane A/J mouse model of lung cancer, Fzd9 mRNA expression is reduced in lung tumors compared to matched, uninvolved lung tissue. Human lung tumors demonstrated reduced Fzd9 mRNA expression compared to matched normal lung tissue. Fzd9 expression is also decreased in human primary dysplastic cell lines, suggesting that loss of Fzd9 expression occurs early in lung tumorigenesis. Non-transforming lung epithelial cell lines (B2B or HBEC) were treated for four weeks with 1% cigarette smoke condensate. B2B cells were treated with the tobacco carcinogen NNK (10μM) for 2 hours. Both treatment conditions resulted in reduced Fzd9 mRNA expression measured by qPCR. Future work will assess Fzd9 expression in an in vitro chemoprevention and smoking model, in tissues from additional mouse models of lung cancer, and in tissues from chemoprevention trials. These initial studies suggest that Fzd9 expression is lost in early smoking-induced lesions. This study has the potential to improve iloprost lung cancer chemoprevention by allowing future trials to more effectively target high-risk patients and by providing a clinical biomarker for identification of chemoprevention candidates. This abstract is also presented as Poster A29. Citation Format: Meredith Tennis, Lori Nield, Dan Merrick, Robert Keith. Frizzled 9 expression is a potential marker of response to iloprost chemoprevention of lung cancer. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr PR03.

10580 Background: Early detection of lung cancer by CT is supported by the National Lung Screening Trial; but while sensitivity of CT is high, specificity is low. Biomarkers to predict the malignant nature of CT detected lung nodules would have great clinical utility. We previously found in a nested case-control study that a 4-target chromosomal aneusomy (CA) FISH panel exhibited sensitivity/specificity of 76%/88% in sputum samples of heavy smokers obtained within 18 months of lung cancer diagnosis. We hypothesized that CA-FISH may be an effective biomarker to assist with clinical decisions in the setting of CT detected lung nodules of indeterminate etiology and attempted to identify a more effective reagent. Methods: Homebrew probes encompassing genomic sequences of EGFR, NXK2-1, PIK3CA, MYC, BRF2, SOX2, PPMID, FGFR1 and the commercial reagent LSI D5S721/D5S23 (Abbott Molecular) were combined in 2-4-target FISH assays to investigate tissue copy number in early stage lung squamous cell carcinoma (SCC, N=19) and adenocarcinoma (AC, N=20). Logistic regression models were used to estimate predictive discrimination [sensitivity, specificity, area under the ROC curve (AUC)]. Results: Copy number gain was largely detected for all markers (mean range 3.39 - PPMID to 4.67 - MYC). Mean copy number of PI3CA, BRF2, SOX2 and FGFR1 were significantly higher in SCC than AC, while NKX2 and MYC were marginally higher in AC than SCC. Gene amplification was detected for all 9 markers, most frequently for SOX2 and FGFR1 (6 and 5 cases) with significant overlap between FGFR1/BRF2 and SOX2/PIK3CA. Based on the AUC results and the existence of targeted inhibitors, the probe set EGFR/FGFR1/MYC/PIK3CA was selected as a candidate for further development as an adjunct to CT screening for early detection. For this selected set,the optimal cutoff based in the linear predictor from logistic regression yields a sensitivity and specificity of 0.85. Conclusions: A highly sensitive/specific CA-FISH probe set was identified which may well complement CT screening in the early diagnosis of lung cancer. This probe set will be tested in the setting of CT detected lung nodules. (Supported by LUNGevity and NCI-Lung Cancer SPORE grants).

The iloprost lung cancer chemoprevention trial is the first to meet a primary endpoint of improvement in endobronchial histology, which is currently considered to be the best intermediate endpoint for chemoprevention (Keith et al, Cancer Prev Res, 2011). This placebo-controlled study offers the opportunity for discovering other surrogate endpoints and predictive biomarkers to incorporate into chemoprevention trials. We compared miRNA expression in follow-up (FU) versus baseline biopsies in relation with histology change or not globally and stratified by treatment arm and smoking status. In the 152 patients randomized in the iloprost trial, the paired baseline and FU biopsies were available in 125 patients: 40/35 current/former smokers in the iloprost arm and 25/25 current/former smokers in the placebo arm, respectively. We planned to analyze 500 biopsies: four biopsies from each of the 125 patients, 2 biopsies at baseline (worst and best diagnosis) and 2 biopsies at FU at the same site after six months of treatment with iloprost or placebo. We selected and analyzed 14 miRNAs differentially expressed during squamous cell lung carcinogenesis in our previous study (Mascaux et al, Eur Resp J, 2009). In total, 496/500 biopsies with appropriate tissue were available. For every biopsy, total RNA was extracted from 8 adjacent 4 um cuts of formalin fixed paraffin embedded (FFPE) adjacent to the diagnostic section. A good reproducibility of the previously published data for the expression changes in the 14 miRNA accross lung preneoplasia stages was shown in the current study in baseline samples. This new study identifies significant changes in miR-34c expression between paired samples when histology is up- or downgraded. miR-34c is significantly down-regulated in paired biopsies when histology is up-graded and inversely. This correlation between miR-34c expression and histology changes is shown including all samples (r spearman correlation=0.23 p=0.0003), but also consistently in subgroups (iloprost.arm, current r=0.26, p=0.041 and former smokers r=0.24, p=0.066, placebo arm, current (r=0.23, p=0.046)). In contrast, miR-9 was significantly down-regulated in follow-up as compared with baseline biopsies, but was not correlated with histology changes. The down-regulation of miR-9 in follow-up versus baseline samples was found in all samples (t test, p In conclusion, miR-34c, a transcriptional target of p53 which is down-regulated by hypermethylation in lung cancer, is down-regulated when histology changes from the normal bronchial mucosa to high-grade bronchial lesions, and inversely, independently of treatment and smoking status. miR-34c expression is a good reflection of baseline histology and histology changes. Alternatively, miR-9 is systematically down-regulated in follow-up biopsies 6 months later at the same site independently of histology, treatment and tobacco status. This suggests that the down-regulation of miR-9 in the follow-up samples may be related to the biopsy: this miRNA could be involved in the tissue repair.

e21137 Background: Thymidylate synthase (TS) catalyzes DNA synthesis and is directly inhibited by pemetrexed, a drug used for treatment of lung cancer. The aim of this study is to analyze the assoc...

7070 Background: Lung cancer is preceded by a premalignant phase during which intervention could decrease associated morbidity and mortality. Molecular characterization of factors involved in controlling progression of bronchial dysplasia (BD) will provide markers of premalignant change and identify targets for chemoprevention. Methods: Immunohistochemical (IHC) analysis of EGFR, HER2, Ki67 and MCM2 expression in BD was undertaken in 268 bronchoscopically obtained biopsies from 134 consecutive subjects recruited to University of Colorado protocols as study subjects with prior sputum atypia and >20 pack years tobacco use or FEV1 < 75% predicted or as never- or healthy-smoker controls. IHC on the worst and best histologic biopsies from each subject were scored and linear regression tests were used to determine significance of correlation between marker expression and histology or proliferation. Results: Analysis of biopsies with the most severe diagnosis from each subject showed a linear relationship between increasing marker expression and severity of dysplastic change for EGFR (p < 0.001), Ki67 (p < 0.001) and MCM2 (p = 0.001) but not HER2 (p = 0.102). Increased expression of either EGFR or HER2 was associated with increased expression of proliferation markers Ki67and MCM2, and combined overexpression of these receptors was associated with the highest levels of proliferation. Finally, in a comparison of subjects that were grouped according to the degree of difference between their best and worst biopsy histology, the absence of an associated trend toward increased EGFR expression with increased difference in histology suggested a field effect in the induction of EGFR expression. Conclusions: Our results 1) demonstrate a direct correlation between levels of EGFR expression and degree of BD, 2) suggest a field effect in the induction of EGFR expression in the airways of subjects with BD and 3) indicate a potential synergistic effect on epithelial proliferation in lesions with increased expression of both EGFR and HER2. These findings suggest that EGFR plays a prominent role in the development and progression of BD and could provide a promising target for chemoprevention of lung cancer. [Table: see text]