Results of a pilot risk-based lung cancer screening study: outcomes and comparisons to a Medicare eligible cohort.

PURPOSE: Risk-based lung cancer screening holds potential to detect more cancers and avert more cancer deaths than screening based on age and smoking history alone, but has not been widely assessed or implemented in the United States. The purpose of this study was to prospectively identify patients for lung cancer screening based on lung cancer risk using the PLCOm2012 model and to compare characteristics, risk profiles, and screening outcomes to a traditionally eligible screening cohort. METHODS: Participants who had a 6 year lung cancer risk score ≥ 1.5% calculated by the PLCOm2012 model and were ineligible for screening under 2015 Medicare guidelines were recruited from a lung cancer screening clinic. After informed consent, participants completed shared decision-making counseling and underwent a low-dose CT (LDCT). Characteristics and screening outcomes of the study population were compared to the traditionally eligible Medicare cohort with Fisher's Exact, t-tests, or Brown Mood tests, as appropriate. RESULTS: From August 2016 to July 2019, the study completed 48 baseline LDCTs. 10% of LDCTs recommended further pulmonary nodule evaluation (Lung-RADs 3 or 4) with two early-stage lung cancers diagnosed in individuals that had quit smoking > 15 years prior. The study population was approximately 5 years older (p = 0.001) and had lower pack years (p = 0.002) than the Medicare cohort. CONCLUSION: Prospective application of risk-based screening identifies screening candidates who are similar to a traditionally eligible Medicare cohort and future research should focus on the impact of risk calculators on lung cancer outcomes and optimal usability in clinical environments. This study was retrospectively registered on clinicaltrials.gov (NCT03683940) on 09/25/2018.

Differences in VA and Non-VA Pulmonary Nodules: All Evaluations Are not Created Equal.

BACKGROUND: Indeterminate pulmonary nodules present a common challenge for clinicians who must recommend surveillance or intervention based on an assessed risk of malignancy. PATIENTS AND METHODS: In this cohort study, patients presenting for indeterminate pulmonary nodule evaluation were enrolled at sites participating in the Colorado SPORE in Lung Cancer. They were followed prospectively and included for analysis if they had a definitive malignant diagnosis, benign diagnosis, or radiographic resolution or stability of their nodule for > 2 years. RESULTS: Patients evaluated at the Veterans Affairs (VA) and non-VA sites were equally as likely to have a malignant diagnosis (48%). The VA cohort represented a higher-risk group than the non-VA cohort regarding smoking history and chronic obstructive pulmonary disease (COPD). There were more squamous cell carcinoma diagnoses among VA malignant nodules (25% vs. 10%) and a later stage at diagnosis among VA patients. Discrimination and calibration of risk calculators produced estimates that were wide-ranging and different when comparing between risk score calculators as well as between VA/non-VA cohorts. Application of current American College of Chest Physicians guidelines to our groups could have resulted in inappropriate resection of 12% of benign nodules. CONCLUSION: Comparison of VA with non-VA patients shows important differences in underlying risk, histology of malignant nodules, and stage at diagnosis. This study highlights the challenge in applying risk calculators to a clinical setting, as the model discrimination and calibration were variable between calculators and between our higher-risk VA and lower-risk non-VA groups. MICROABSTRACT: Risk stratification and management of indeterminate pulmonary nodules (IPNs) is a common clinical problem. In this prospective cohort study of 282 patients with IPNs from Veterans Affairs (VA) and non-VA sites, we found differences in patient and nodule characteristics, histology and diagnostic stage, and risk calculator performance. Our findings highlight challenges and shortcomings of current IPN management guidelines and tools.

Determinants Associated With Longitudinal Adherence to Annual Lung Cancer Screening: A Retrospective Analysis of Claims Data.

OBJECTIVE: Lung cancer screening (LCS) efficacy is highly dependent on adherence to annual screening, but little is known about real-world adherence determinants. We used insurance claims data to examine associations between LCS annual adherence and demographic, comorbidity, health care usage, and geographic factors. MATERIALS AND METHODS: Insurance claims data for all individuals with an LCS low-dose CT scan were obtained from the Colorado All Payer Claims Dataset. Adherence was defined as a second claim for a screening CT 10 to 18 months after the index claim. Cox proportional hazards regression was used to define the relationship between annual adherence and age, gender, insurance type, residence location, outpatient health care usage, and comorbidity burden. RESULTS: After exclusions, the final data set consisted of 9,056 records with 3,072 adherent, 3,570 nonadherent, and 2,414 censored (unclassifiable) individuals. Less adherence was associated with ages 55 to 59 (hazard ratio [HR] = 0.80, 99% confidence interval [CI] = 0.67-0.94), 60 to 64 (HR = 0.83, 99% CI = 0.71-0.97), and 75 to 79 (HR = 0.79, 99% CI = 0.65-0.97); rural residence (HR = 0.56, 99% CI = 0.43-0.73); Medicare fee-for-service (HR = 0.45, 99% CI = 0.39-0.51), and Medicaid (HR = 0.50, 99% CI = 0.40-0.62). A significant interaction between outpatient health care usage and comorbidity was also observed. Increased outpatient usage was associated with increased adherence and was most pronounced for individuals without comorbidities. CONCLUSIONS: This population-based description of LCS adherence determinants provides insight into populations that might benefit from specific interventions targeted toward improving adherence and maximizing LCS benefit. Quantifying population-based adherence rates and understanding factors associated with annual adherence are critical to improving screening adherence and reducing lung cancer death.

Development of syngeneic murine cell lines for use in immunocompetent orthotopic lung cancer models.

BACKGROUND: Immunocompetent animal models are required to study tumor-host interactions, immunotherapy, and immunotherapeutic combinations, however the currently available immunocompetent lung cancer models have substantial limitations. While orthotopic models potentially help fill this gap, the utility of these models has been limited by the very small number of murine lung cancer cell lines capable of forming orthotopic tumors in immunocompetent C57BL/6 hosts. METHODS: Primary lung tumors with specific genetic alterations were created in C57BL/6 background mice. These tumors were then passaged through other animals to increase tumorigenicity and select for the ability to grow in a non-self animal. Once tumors demonstrated growth in a non-self host, cell lines were established. Successful cell lines were evaluated for the ability to produce orthotopic lung tumors in immunocompetent hosts. RESULTS: We produced six murine lung cancer lines capable of orthotopic lung tumor formation in immunocompetent C57BL/6 animals. These lines demonstrate the expected genetic alterations based on their primary tumor genetics. CONCLUSIONS: These novel cell lines will be useful for evaluating tumor-host interactions, the impact of specific oncogenic alterations on the tumor microenvironment, and immunotherapeutic approaches. This method of generating murine lines capable of orthotopic growth can likely be applied to other tumors and will broaden the applicability of pre-clinical testing of immunotherapeutic treatment regimens.

Impact of a Hybrid Lung Cancer Screening Model on Patient Outcomes and Provider Behavior.

BACKGROUND: Lung cancer screening (LCS) implementation is complicated by the Centers for Medicare and Medicaid Services reimbursement requirements of shared decision-making and tobacco cessation counseling. LCS programs can utilize different structures to meet these requirements, but the impact of programmatic structure on provider behavior and screening outcomes is poorly described. PATIENTS AND METHODS: In a retrospective chart review of 624 patients in a hybrid structure, academic LCS program, we compared characteristics and outcomes of primary care provider (PCP)- and specialist-screened patients. We also assessed the impact of the availability of an LCS specialty clinic and best practice advisory (BPA) on PCP ordering patterns using electronic medical record generated reports. RESULTS: During the study period of July 1, 2014 through June 30, 2018, 48% of patients were specialist-screened and 52% were PCP-screened; there were no clinically relevant differences in patient characteristics or screening outcomes between these populations. PCPs demonstrate distinct practice patterns when offered the choice of specialist-driven or PCP-driven screening. Increased exposure to a LCS BPA is associated with increased PCP screening orders. The addition of a nurse navigator into the LCS program increased documentation of shared decision-making and tobacco cessation counseling to > 95% and virtually eliminated screening of ineligible patients. CONCLUSIONS: Systematic interventions including a BPA and nurse navigator are associated with increased screening and improved program quality, as evidenced by reduced screening of ineligible patients, increased lung cancer risk of the screened population, and improved compliance with LCS guidelines. Individual PCPs demonstrate clear preferences regarding LCS that should be considered in program design.

Incorporating Coexisting Chronic Illness into Decisions about Patient Selection for Lung Cancer Screening. An Official American Thoracic Society Research Statement.

BACKGROUND: Lung cancer screening (LCS) has the potential to reduce the risk of lung cancer death in healthy individuals, but the impact of coexisting chronic illnesses on LCS outcomes has not been well defined. Consideration of the complex relationship between baseline risk of lung cancer, treatment-related harms, and risk of death from competing causes is crucial in determining the balance of benefits and harms of LCS. OBJECTIVES: To summarize evidence, identify knowledge and research gaps, prioritize topics, and propose methods for future research on how best to incorporate comorbidities in making decisions regarding LCS. METHODS: A multidisciplinary group of international clinicians and researchers reviewed available data on the effects of comorbidities on LCS outcomes, focusing on the juxtaposition of lung cancer risk and competing risks of death, consideration of benefits and risks in patients with chronic obstructive pulmonary disease, communication of risk, and treatment of screen-detected lung cancer. RESULTS: This statement identifies gaps in knowledge regarding how comorbidities and competing causes of death impact outcomes in LCS, and we have developed questions to help guide future research efforts to better inform patient selection, education, and implementation of LCS. CONCLUSIONS: There is an urgent need for further research that can help guide clinical decision-making with patients who may not benefit from LCS owing to coexisting chronic illness. This statement establishes a research framework to address essential questions regarding how to incorporate and communicate risks of comorbidities into patient selection and decisions regarding LCS.

Adenoviral vectors transduce alveolar macrophages in lung cancer models.

Adenoviral vectors expressing Cre recombinase are commonly used to initiate tumor formation in murine lung cancer models. While these vectors are designed to target genetic recombination to lung epithelial cells, adenoviruses can infect additional cell types that potentially influence tumor development. Our goal was to explore the consequences of adenoviral-mediated alveolar macrophage (AM) transduction in a Kras-initiated lung tumor model. As expected, treatment of animals harboring the KrasLSL-G12D allele and an inducible green fluorescence protein (GFP) tracking allele with an adenoviral vector expressing Cre recombinase under the control of the cytomegalovirus (CMV) promoter (Ad5-CMV-Cre), caused GFP-positive lung adenocarcinomas. Surprisingly, however, up to 70% of the total GFP+ cells were AM, and GFP+ AM could be detected 6 months after tumor initiation, and transduced AM demonstrated Kras activation and increased proliferation. In contrast, recombination was not detected in other immune cell populations and AM recombination could be eliminated by tumor initiation with an adenovirus expressing Cre recombinase under the control of the surfactant protein C (SPC) promoter. In addition, AM isolated from KrasLSL-G12D animals and transduced by Ad5-CMV-Cre ex vivo displayed prolonged survival in vitro and increased the growth of murine lung adenocarcinoma CMT/167 cells when co-injected in an orthotopic flank model. Given the importance of the immune system in tumor development and progression, inadvertent AM transduction by Ad5-CMV-Cre merits careful consideration during lung cancer model selection particularly if studies evaluating the tumor-immune interactions are planned.

Complement Activation via a C3a Receptor Pathway Alters CD4+ T Lymphocytes and Mediates Lung Cancer Progression.

The complement cascade is a part of the innate immune system that acts primarily to remove pathogens and injured cells. However, complement activation is also peculiarly associated with tumor progression. Here we report mechanistic insights into this association in multiple immunocompetent orthotopic models of lung cancer. After tumor engraftment, we observed systemic activation of the complement cascade as reflected by elevated levels of the key regulator C3a. Notably, growth of primary tumors and metastases was both strongly inhibited in C3-deficient mice (C3-/- mice), with tumors undetectable in many subjects. Growth inhibition was associated with increased numbers of IFNγ+/TNFα+/IL10+ CD4+ and CD8+ T cells. Immunodepletion of CD4+ but not CD8+ T cells in tumor-bearing subjects reversed the inhibitory effects of C3 deletion. Similarly, antagonists of the C3a or C5a receptors inhibited tumor growth. Investigations using multiple tumor cell lines in the orthotopic model suggested the involvement of a C3/C3 receptor autocrine signaling loop in regulating tumor growth. Overall, our findings offer functional evidence that complement activation serves as a critical immunomodulator in lung cancer progression, acting to drive immune escape via a C3/C5-dependent pathway.Significance: This provocative study suggests that inhibiting complement activation may heighten immunotherapeutic responses in lung cancer, offering findings with immediate implications, given the existing clinical availability of complement antagonists. Cancer Res; 78(1); 143-56. ©2017 AACR.

Reduced Smad4 expression and DNA topoisomerase inhibitor chemosensitivity in non-small cell lung cancer.

OBJECTIVE: Smad4 is a tumor suppressor that transduces transforming growth factor beta signaling and regulates genomic stability. We previously found that Smad4 knockdown in vitro inhibited DNA repair and increased sensitivity to DNA topoisomerase inhibitors. In this study, we assessed the association between reduced Smad4 expression and DNA topoisomerase inhibitor sensitivity in human non-small cell lung cancer (NSCLC) patients and evaluated the relationship between genomic alterations of Smad4 and molecular alterations in DNA repair molecules. MATERIALS AND METHODS: We retrospectively identified NSCLC patients who received etoposide or gemcitabine. Chemotherapeutic response was quantified by RECIST 1.1 criteria and Smad4 expression was assessed by immunohistochemistry. Relationships between Smad4 mutation and DNA repair molecule mutations were evaluated using publically available datasets. RESULTS: We identified 28 individuals who received 30 treatments with gemcitabine or etoposide containing regimens for NSCLC. Reduced Smad4 expression was seen in 13/28 patients and was not associated with significant differences in clinical or pathologic parameters. Patients with reduced Smad4 expression had a larger response to DNA topoisomerase inhibitor containing regimens then patients with high Smad4 expression (-25.7% vs. -6.8% in lesion size, p=0.03); this relationship was more pronounced with gemcitabine containing regimens. The overall treatment response was higher in patients with reduced Smad4 expression (8/14 vs 2/16 p=0.02). Analysis of data from The Cancer Genome Atlas revealed that Smad4 mutation or homozygous loss was mutually exclusive with genomic alterations in DNA repair molecules. CONCLUSIONS: Reduced Smad4 expression may predict responsiveness to regimens that contain DNA topoisomerase inhibitors. That Smad4 signaling alterations are mutually exclusive with alterations in DNA repair machinery is consistent with an important role of Smad4 in regulating DNA repair.

Smad4 loss promotes lung cancer formation but increases sensitivity to DNA topoisomerase inhibitors.

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.

A randomized, double-blind pilot study of dexmedetomidine versus midazolam for intensive care unit sedation: patient recall of their experiences and short-term psychological outcomes.

INTRODUCTION: Sedation with dexmedetomidine may facilitate ventilator liberation and limit the occurrence of delirium. No trial has assessed patient recall or the development of psychological outcomes after dexmedetomidine sedation. This pilot study evaluated whether transitioning benzodiazepine sedation to dexmedetomidine alters patient recall and the incidence of anxiety, depression, or acute stress disorder (ASD). METHODS: This investigation was a randomized, double-blind, single-center study. Existing continuous benzodiazepine sedation was converted to dexmedetomidine or midazolam when patients qualified for daily awakenings. Sedation was titrated to achieve Riker sedation agitation scores of 3 to 4. The intensive care unit (ICU) Stressful Experiences Questionnaire, hospital anxiety and depression scale, and the impact of event scale-revised were administered before hospital discharge to assess recall, anxiety, depression, and manifestations of ASD. RESULTS: A total of 11 patients received dexmedetomidine, and 12 patients received midazolam. Median dosing was 0.61 µg/kg/h for 3.5 days for dexmedetomidine and 3.7 mg/h for 3 days for midazolam. Attainment of goal sedation and analgesia was similar; however, more dexmedetomidine patients experienced agitation and pain. The median duration of mechanical ventilation from study drug initiation to extubation was 3.4 days in dexmedetomidine patients and 2.9 days in midazolam patients. Dexmedetomidine patients remembered 18.5 experiences compared with 8.5 in midazolam patients (P = .015). Rates of anxiety and depression were similar. In all, 5 (62.5%) dexmedetomidine patients and 1 (12.5%) midazolam patient manifested ASD (P = .063), and 1 dexmedetomidine patient and 5 midazolam patients developed new-onset delirium (P = .07). Hypotension occurred in 10 (90.9%) dexmedotomidine patients and 6 (50%) midazolam patients (P = .069). CONCLUSIONS: Transitioning benzodiazepine sedation to dexmedetomidine when patients qualify for daily awakenings may reduce the development of delirium and facilitate remembrance of ICU experiences but may lead to manifestations of ASD. Monitoring hypotension is required for both the sedatives. Additional comparative studies focusing on the long-term impact of ICU recall and psychological outcomes are needed.

Role of PTEN in basal cell derived lung carcinogenesis.

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 ß receptor type II (TGFßRII) 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.

A randomized, double-blind, placebo-controlled dose range study of dexmedetomidine as adjunctive therapy for alcohol withdrawal.

OBJECTIVES: To evaluate dexmedetomidine as adjunctive therapy to lorazepam for severe alcohol withdrawal. DESIGN: Prospective, randomized, double-blind, placebo-controlled trial. SETTING: Single center; medical ICU. PATIENTS: Twenty-four adult patients with a Clinical Institute Withdrawal Assessment score greater than or equal to 15 despite greater than or equal to 16 mg of lorazepam over a 4-hour period. INTERVENTIONS: Patients received a symptom-triggered Clinical Institute Withdrawal Assessment protocol with lorazepam and were randomized to dexmedetomidine 1.2 µg/kg/hr (high dose), 0.4 µg/kg/hr (low dose), or placebo as adjunctive therapy for up to 5 days or resolution of withdrawal symptoms. MEASUREMENT AND MAIN RESULTS: High-dose and low-dose groups were combined as a single dexmedetomidine group for primary analysis with secondary analysis exploring a dose-response relationship. The difference in 24-hour lorazepam requirements after versus before study drug was greater in the dexmedetomidine group compared with the placebo group (-56 mg vs -8 mg, p = 0.037). Median differences were similar for high dose and low dose. The 7-day cumulative lorazepam requirements were not statistically different between dexmedetomidine and placebo (159 mg vs 181 mg). Clinical Institute Withdrawal Assessment or Riker sedation-agitation scale scores representing severe agitation (13% vs 25%) or moderate agitation (27% vs 22%) within 24 hours of initiating study drug were similar for dexmedetomidine and placebo groups, respectively. Bradycardia occurred more frequently in the dexmedetomidine group versus placebo group (25% vs 0%, p = not significant), with the majority of bradycardia occurring in the high-dose group (37.5%). Study drug rate adjustments occurred more often in the dexmedetomidine group compared with the placebo group (50% vs 0%, p = 0.02). Neither endotracheal intubation nor seizure occurred in any group while on study drug. CONCLUSIONS: Adjunctive dexmedetomidine for severe alcohol withdrawal maintains symptom control and reduces lorazepam exposure in the short term, but not long term, when using a symptom-triggered protocol. Monitoring for bradycardia is needed with dexmedetomidine but the occurrence may be lessened with low dose. Further study is needed to evaluate the clinical impact of dexmedetomidine.

Loss of transforming growth factor beta type II receptor increases aggressive tumor behavior and reduces survival in lung adenocarcinoma and squamous cell carcinoma.

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 TGFß type II receptor (TGFßRII) promotes lung adenocarcinoma and SCC carcinogenesis. EXPERIMENTAL DESIGN: We examined TGFßRII expression at the protein and mRNA levels in human NSCLC samples and assessed the relationship between TGFßRII expression and clinicopathologic parameters. To determine whether sporadic TGFßRII deletion in airway epithelial cells induces NSCLC formation, we targeted TGFßRII deletion alone and in combination with oncogenic Kras(G12D) to murine airways using a keratin 5 (K5) promoter and inducible Cre recombinase. RESULTS: Reduced TGFßRII 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 TGFßRII deletion in mouse airway epithelia increases the size and number of Kras(G12D)-initiated adenocarcinoma and SCC. TGFßRII deletion increases proliferation, local inflammation, and TGFß ligand elaboration; TGFßRII knockdown in airway epithelial cells increases migration and invasion. CONCLUSIONS: Reduced TGFßRII expression in human NSCLC is associated with more aggressive tumor behavior and inflammation that is, at least partially, mediated by increased TGFß1 expression. TGFßRII deletion in mouse airway epithelial cells promotes adenocarcinoma and SCC formation, indicating that TGFßRII loss plays a causal role in lung carcinogenesis. That TGFßRII shows haploid insufficiency suggests that a 50% TGFßRII protein reduction would negatively impact lung cancer prognosis.

Two sides of the story? Smad4 loss in pancreatic cancer versus head-and-neck cancer.

TGFß signaling Smads (Smad2, 3, and 4) were suspected tumor suppressors soon after their discovery. Nearly two decades of research confirmed this role and revealed other divergent and cancer-specific functions including paradoxical tumor promotion effects. Although Smad4 is the most potent tumor suppressor, its functions are highly context-specific as exemplified by pancreatic cancer and head-and-neck cancer: in pancreatic cancer, Smad4 loss cannot initiate tumor formation but promotes metastases while in head-and-neck cancer Smad4 loss promotes cancer progression but also initiates tumor formation, likely through effects on genomic instability. The differing consequences of impaired Smad signaling in human cancers and the molecular mechanisms that underpin these differences will have important implications for the design and application of novel targeted therapies.

N-nitroso-tris-chloroethylurea induces premalignant squamous dysplasia in mice.

Squamous cell carcinoma (SCC) and premalignant endobronchial lesions have been difficult to study in murine models. In this study, we evaluate the topical N-nitroso-tris-chloroethylurea (NTCU) murine SCC model, determine the extent to which resulting premalignant airway dysplasia develops, discuss clinicopathologic grading criteria in lesion progression, and confirm that immunohistochemical (IHC) staining patterns are consistent with those observed in human endobronchial dysplasia and SCC. Male and female FVB mice were treated biweekly with topical NTCU (4, 8, or 40 mmol/L) or vehicle for 32 weeks. Following sacrifice, squamous cell lesions were enumerated and categorized into the following groups: flat atypia, low-grade dysplasia, high-grade dysplasia, and invasive SCC. The 40 mmol/L NTCU concentration produced the entire spectrum of premalignant dysplasias and squamous cell carcinomas, but was associated with poor survival. Concentrations of 4 and 8 mmol/L NTCU were better tolerated and produced only significant levels of flat atypia. Squamous origin of the range of observed lesions was confirmed with IHC staining for cytokeratin 5/6, p63, thyroid transcription factor-1 (TTF-1), and Napsin-A. This study shows that topical application of high-dose NTCU produces endobronchial premalignant lesions with classic squamous characteristics and should allow for improved preclinical evaluation of potential chemopreventive agents.

Tobacco-specific carcinogen nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces AKT activation in head and neck epithelia.

Exposure to tobacco carcinogens is causally associated with head and neck squamous cell carcinoma (HNSCC), but the underlying molecular mechanisms remain unclear. Here, we reported that AKT is activated at a higher frequency in both HNSCC tumors and the adjacent mucosa from HNSCC patients who are smokers than those from HNSCC patients who are non-smokers. Adding physiologically relevant concentrations of 4-(methylnitrosamino)-1-(3-pyridyl)-1-1butanone (NNK), a major tobacco carcinogen, to normal head and neck epithelial cells and HNSCC cell lines, rapidly and constitutively activated AKT through phosphorylation in a dose- and time-dependent manner. AKT phosphorylation was associated with activation of downstream signaling mediators BAD, MDM2, GSK-3ß, mTOR. These alterations correlated with increased proliferation and decreased etoposide-induced apoptosis in NNK-exposed cells. Finally, NNK exposure to mouse head and neck epithelia resulted in epithelial hyperproliferation and reduced apoptosis, which is correlated with AKT activation. Our results suggest that AKT activation is an early event and plays a pivotal role in mediating tobacco-induced HNSCC carcinogenesis.