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The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. Hamp;E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

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.

PDF file - 1.3MB, H/E (A) and IHC staining of adenocarcinoma with CK5/6 (B), p63 (C), TTF-1 (D), and NAPSIN-A (E). magnification: 400X.

Bronchial dysplasia (BD), a presumed precursor of pulmonary squamous cell carcinoma (SCC), rarely progresses to invasive cancer. A high-risk cohort at the University of Colorado provided an opportunity to directly sample airway epithelium at mapped sites on successive bronchoscopies. We have hypothesized that persistent dysplastic lesions showing a similar or higher level of dysplasia on follow-up biopsy, are associated with increased risk for the development of SCC. Endoscopic biopsies from 188 high-risk subjects were histologically classified according to the current WHO classification for BD using a numeric histology score ranging from 1 to 8 representing normal bronchial mucosa through invasive lung cancer. Differences in follow-up histology scores were compared between sites classified by clinical, histologic, and immunohistochemical variables. Subjects with a higher frequency of sites that persist or progress to high-grade dysplasia (≥37.5% persist/progress, N = 35 versus N = 114) show a significant association with development of incident invasive SCC (adjusted HR, 7.84; 95% confidence interval, 1.56–39.39), and those with incident lung SCC have adjusted mean follow-up histology scores 1.55 U higher than in subjects without lung cancer. Current smoking, elevated Ki67 growth fraction, histologic features of angiogenic squamous dysplasia (ASD) and higher histology score in baseline biopsies are significantly associated with increased follow-up histology scores. These results show that persistent BD is associated with the development of invasive SCC. Furthermore, increased expression of Ki67, the presence of angiogenic change and degree of baseline atypia are associated with persistence of BD. Cancer Prev Res; 9(1); 96–104. ©2015 AACR.

Adverse Events Summary

PDF file - 73K, Association of miRNA expression at BL with response. Legend: This table reports the three miRNAs (miR-34c, miR-224, miR-375) that are significantly differentially expressed at BL in responders as compared to non-responders in at least one comparison The table shows up- or down-regulation in responders compared to non-responders. The significant p-values from Welch's t-test are reported. "FDR" indicates that the p-value is significant after adjustment for False Discovery Rate (FDR). Odds ratios (OR) were calculated by logistic regression model and reported with their 95% confidence interval and p-value. When either the t-test or the OR was significant, both are reported.

Supplementary Figure S1 from Oral Iloprost Improves Endobronchial Dysplasia in Former Smokers

Chromosomal instability is central to the process of carcinogenesis. The genome-wide detection of somatic chromosomal alterations (SCA) in small premalignant lesions remains challenging because sample heterogeneity dilutes the aberrant cell information. To overcome this hurdle, we focused on the B allele frequency data from single-nucleotide polymorphism microarrays (SNP arrays). The difference of allelic fractions between paired tumor and normal samples from the same patient (delta-θ) provides a simple but sensitive detection of SCA in the affected tissue. We applied the delta-θ approach to small, heterogeneous clinical specimens, including endobronchial biopsies and brushings. Regions identified by delta-θ were validated by FISH and quantitative PCR in heterogeneous samples. Distinctive genomic variations were successfully detected across the whole genome in all invasive cancer cases (6 of 6), carcinoma in situ (3 of 3), and high-grade dysplasia (severe or moderate; 3 of 11). Not only well-described SCAs in lung squamous cell carcinoma, but also several novel chromosomal alterations were frequently found across the preinvasive dysplastic cases. Within these novel regions, losses of putative tumor suppressors (RNF20 and SSBP2) and an amplification of RASGRP3 gene with oncogenic activity were observed. Widespread sampling of the airway during bronchoscopy demonstrated that field cancerization reflected by SCAs at multiple sites was detectable. SNP arrays combined with delta-θ analysis can detect SCAs in heterogeneous clinical sample and expand our ability to assess genomic instability in the airway epithelium as a biomarker of lung cancer risk. Cancer Prev Res; 7(2); 255–65. ©2013 AACR.

XLS - 100K, The list of SCAs detected by delta-theta in clinical specimens.

Supplementary Table S1 from Oral Iloprost Improves Endobronchial Dysplasia in Former Smokers

PDF file - 69K

Legends for Supplementary Tables 1 and 2

PDF - 85K, Different concepts of delta-theta and modified BAF.

PDF file - 36K, Distribution of histologies at BL and at FU in pairs of samples with best or worse histology at baseline. Legend : N=number, BL=baseline, FU=follow-up

Supplemental figure S2. Univariable analysis comparing frequency of SCC in cases grouped by highest baseline (BL) histology score (HS) or mean baseline histology score using a cutoff of HS > 5 (moderate dysplasia).

PDF file - 57K, Distribution of histological responders and non-responders in iloprost and in placebo arm Legend :"Worst histology score criterion" = response is defined as a decrease of 1 or more in worst histology score of all biopsies from the same patients after treatment as compared to before, as used in the iloprost chemoprevention trial (11); "paired sample based criteria" = response is defined by a decrease of one histological grade within matched paired biopsies taken at the same site before and after treatment.

SUPPLEMENTAL TABLE S1. BIOPSY AND CLINCAL CHARACTERISTICS. SUPPLEMENTAL TABLE S2. BASELINE HISTOLOGY AND OUTCOME.

PDF file - 99K, The number of animals in each score category from the behavioral index scoring system in the 4mM and 8mM treatment groups.

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.

PDF file - 37K, Distribution of histologies at BL and FU between the treatment arms and stratified by smoking status Legend: N=number, pts=patients

PDF file - 24K, Differences in lesion number between the males and females of the 40mM cohort.

PDF - 138K, The comparisons of representative delta-θ signals between 2 different methods of DNA isolation from brushing.

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.

PDF - 212K, Histology grade of overall biopsies in individual patients with preinvasive lesions.

Perspective on This Article from Oral Iloprost Improves Endobronchial Dysplasia in Former Smokers

PDF - 240K, Overall segmentation results across the entire autosomal chromosomes in the genomic DNA titration series.

PDF - 79K, Legends for Supplementary Figures 1 through 5

PDF - 494K, Detection and validation of SCAs in a heterogeneous cancer case by FISH assays.

PDF file - 152K, weight fluctuation in animals receiving 40mM NTCU. This group was divided into two waves (panels A and B), with differing periods of suspensions (arrows) throughout the study. Weight recovery is observable following treatment suspensions. Cessation of treatment is represented with an arrowhead.

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.

ALK and ROS1 patient specific data

Supplementary Figures 1-5

Custom capture-based NGS panel of 48 genes

Purpose: Despite initial benefit from tyrosine kinase inhibitors (TKIs), patients with advanced non–small cell lung cancer (NSCLC) harboring ALK (ALK+) and ROS1 (ROS1+) gene fusions ultimately progress. Here, we report on the potential resistance mechanisms in a series of patients with ALK+ and ROS1+ NSCLC progressing on different types and/or lines of ROS1/ALK–targeted therapy.Experimental Design: We used a combination of next-generation sequencing (NGS), multiplex mutation assay, direct DNA sequencing, RT-PCR, and FISH to identify fusion variants/partners and copy-number gain (CNG), kinase domain mutations (KDM), and copy-number variations (CNVs) in other cancer-related genes. We performed testing on 12 ROS1+ and 43 ALK+ patients.Results: One of 12 ROS1+ (8%) and 15 of 43 (35%) ALK+ patients harbored KDM. In the ROS1+ cohort, we identified KIT and β-catenin mutations and HER2-mediated bypass signaling as non-ROS1–dominant resistance mechanisms. In the ALK+ cohort, we identified a novel NRG1 gene fusion, a RET fusion, 2 EGFR, and 3 KRAS mutations, as well as mutations in IDH1, RIT1, NOTCH, and NF1. In addition, we identified CNV in multiple proto-oncogenes genes including PDGFRA, KIT, KDR, GNAS, K/HRAS, RET, NTRK1, MAP2K1, and others.Conclusions: We identified a putative TKI resistance mechanism in six of 12 (50%) ROS1+ patients and 37 of 43 (86%) ALK+ patients. Our data suggest that a focus on KDMs will miss most resistance mechanisms; broader gene testing strategies and functional validation is warranted to devise new therapeutic strategies for drug resistance. Clin Cancer Res; 24(14); 3334–47. ©2018 AACR.

Supplemental table S1 documents comparability between final groupings of baseline biopsies used for gene expression comparisons between persistent and regressive sites demonstrating comparability in all key histologic and clinical variables with the exception of higher inflammation in the regressive group. Supplemental table S2 describes the number of differentially expressed genes that distinguish the four initial groups of bronchial biopsies including persistent bronchial dysplasia (BD), regressive BD, progressive non-dysplasia (ND) and stable ND. The number of genes differentially expressed between the groups highlight the finding that differences are greater in respect to the outcome of the lesions at a given site versus the degree of dysplasia in the baseline biopsy. Most notably the difference between regressive BD and stable ND, two groups with histologically distinct baseline biopsies show fewer differentially expressed genes than all but one other group comparison including the histologically indistinguishable persistent and regressive BD groups. These findings are consistent with persistent and regressive sites representing biologically distinct lesions. Supplemental table S3 shows airway locations, temporal histologic features of full airways and clinical characteristics associated with each specimen used for gene expression analyses. The table is arranged by persistent versus regressive status and presents specimens in descending order of baseline histologic score within each group. The specimen numbers correspond to those notated in figure 1B of the manuscript. Supplemental figure S1 shows a heat map using differentially expressed genes to demonstrate the rationale for combining groups in the final comparison. The heatmap demonstrates that persistent BD and progressive ND sites, which are combined to represent persistent sites, show similar gene expression profiles, with only cases Group3_051, Group3_086, Group3_113 and Group4_070 showing profiles that demonstrate similarity to the regressive BD/stable ND group. The regressive BD and stable ND, which are combined to represent regressive sites, also show similar gene expression profiles that are opposite to those seen in the persistent site group with only cases Group2_037, Group2_085 and Group2_092 showing profiles that demonstrate similarity to the persistent BD/progressive ND group. Supplemental tables S4 - S6 present full data genelists for genes associated with pathways showing significantly altered activity in comparisons of persistent and regressive BD (supplemental table S3), genes associated with pathways that are altered in relation to increasing histologic score (supplemental table S4) and upstream regulators related to persistence and histologic score (supplemental table S5). Supplemental table S7 shows documents comparability between final groupings of baseline biopsies used for analyses of the composition of inflammatory infiltrates in comparisons between a validational set of persistent and regressive sites. Comparability is demonstrated in all key histologic and clinical variables including mean inflammation score and percent of high inflammation score biopsies between the two groups. A higher mean baseline histologic score was seen in the persistent group. Supplemental figure S2 shows subset analyses of the validational set of persistent and regressive sites with comparison of these groups by tissue compartment (epithelial and stromal) and overall degree of inflammation (supplemental figures S2A and S2B). Supplemental figure S2C shows the changes in expression of genes from the original gene expression array analysis data for those genes associated with polarization of subsets of inflammatory cells. The genes with significant or near significant change provide evidence that the activity of certain subsets of macrophages and T-lymphocytes correlate with persistence or regression of BD. These subsets are further studied in figure 5 of the main text.

Read depths for ALK and ROS1 used in NGS

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates anin vitromodel of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticatedex vivomodels and a foundation for the study of carcinogenesis and chemoprevention strategies.

Lung cancers bearing oncogenically-mutated EGFR represent a significant fraction of lung adenocarcinomas (LUADs) for which EGFR-targeting tyrosine kinase inhibitors (TKIs) provide a highly effective therapeutic approach. However, these lung cancers eventually acquire resistance and undergo progression within a characteristically broad treatment duration range. Our previous study of EGFR mutant lung cancer biopsies highlighted the positive association of a TKI-induced interferon γ transcriptional response with increased time to treatment progression. To test the hypothesis that host immunity contributes to the TKI response, we developed novel genetically-engineered mouse models of EGFR mutant lung cancer bearing exon 19 deletions (del19) or the L860R missense mutation. Both oncogenic EGFR mouse models developed multifocal LUADs from which transplantable cancer cell lines sensitive to the EGFR-specific TKIs, gefitinib and osimertinib, were derived. When propagated orthotopically in the left lungs of syngeneic C57BL/6 mice, deep and durable shrinkage of the cell line-derived tumors was observed in response to daily treatment with osimertinib. By contrast, orthotopic tumors propagated in immune deficientnu/numice exhibited modest tumor shrinkage followed by rapid progression on continuous osimertinib treatment. Importantly, osimertinib treatment significantly increased intratumoral CD3+ T cell content relative to diluent treatment. The findings provide strong evidence supporting the requirement for adaptive immunity in the durable therapeutic control of EGFR mutant lung cancer.

Chest tubes account for a large proportion of postoperative pain after cardiothoracic operations. The objective of this study was to develop a novel, cost-effective, easy-to-use, lidocaine-eluting coating to reduce pain associated with postoperative chest tubes. A lidocaine-eluting hydrogel was developed by dispersing lidocaine-loaded nanoparticles in an aqueous solution containing gelatin (5%). Glutaraldehyde (1%) was added to crosslink the gelatin into a hydrogel. The hydrogel was dehydrated, resulting in a thin, stable polymer. Sterile lidocaine hydrogel-coated silicone discs and control discs were prepared and surgically implanted in the subcutaneous space of C57B6 mice. Using von Frey filaments, mice underwent preoperative baseline pain testing, followed by pain testing on post-procedure day 1 and 3. On post-procedure day 1, mice implanted with control discs demonstrated no change in pain tolerance compared to baseline, while mice implanted with 20 mg and 80 mg lidocaine-loaded discs demonstrated a 2.4-fold (P = 0.36) and 4.7-fold (P = 0.01) increase in pain tolerance, respectively. On post-procedure day 3, mice implanted with control discs demonstrated a 0.7-fold decrease in pain tolerance compared to baseline, while mice implanted with 20 mg and 80 mg lidocaine-loaded discs demonstrated a 1.8-fold (P = 0.88) and 8.4-fold (P = 0.02) increase in pain tolerance, respectively. Our results demonstrate successful development of a lidocaine-eluting chest tube with hydrogel coating, leading to improved pain tolerance in vivo. The concept of a drug-eluting drain coating has significant importance due to its potential universal application in a variety of drain types and insertion locations.

A pulmonary cyst usually refers to an air-filled space with a smooth, thin wall. Fluid-filled cystic lesions of the lungs include a range of etiologies such as true cysts, congenital malformations, infections, and benign and malignant neoplasms. With relatively little solid component, these lesions often have similar imaging appearances to one another. This article focuses on key imaging features and clinical characteristics that can be used to narrow the differential diagnosis.

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.

Despite the success of immune checkpoint blockade therapy, few strategies sufficiently overcome immunosuppression within the tumor microenvironment (TME). Targeting regulatory T cells (T(regs)) is challenging, because perturbing intratumoral T(reg) function must be specific enough to avoid systemic inflammatory side effects. Thus, no T(reg)-targeted agents have proven both safe and efficacious in patients with cancer. Neuropilin-1 (NRP1) is recognized for its role in supporting intratumoral T(reg) function while being dispensable for peripheral homeostasis. Nonetheless, little is known about the biology of human NRP1 T(regs) and the signals that regulate NRP1 expression. Here, we report that NRP1 is preferentially expressed on intratumoral T(regs) across six distinct cancer types compared to healthy donor peripheral blood [peripheral blood lymphocyte (PBL)] and site-matched, noncancer tissue. Furthermore, NRP1(+) T(reg) prevalence is associated with reduced progression-free survival in head and neck cancer. Human NRP1(+) T(regs) have broad activation programs and elevated suppressive function. Unlike mouse T(regs), we demonstrate that NRP1 identifies a transient activation state of human T(regs) driven by continuous T cell receptor (TCR) signaling through the mitogen-activated protein kinase pathway and interleukin-2 exposure. The prevalence of NRP1(+) T(regs) in patient PBL correlates with the intratumoral abundance of NRP1(+) T(regs) and may indicate higher disease burden. These findings support further clinical evaluation of NRP1 as a suitable therapeutic target to enhance antitumor immunity by inhibiting T(reg) function in the TME.

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.

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