Inhibition of dUTPase induces synthetic lethality with thymidylate synthase-targeted therapies in non-small cell lung cancer

Chemotherapies that target thymidylate synthase (TS) continue to see considerable clinical expansion in non-small cell lung cancer (NSCLC). One drawback to TS-targeted therapies is drug resistance and subsequent treatment failure. Novel therapeutic and biomarker-driven strategies are urgently needed. The enzyme deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is reported to protect tumor cells from aberrant misincorporation of uracil during TS inhibition. The goal of this study was to investigate the expression and significance of dUTPase in mediating response to TS-targeted agents in NSCLC. The expression of dUTPase in NSCLC cell lines and clinical specimens was measured by quantitative real-time reverse transcriptase PCR and immunohistochemistry. Using a validated RNA interference approach, dUTPase was effectively silenced in a panel of NSCLC cell lines and response to the fluoropyrimidine fluorodeoxyuridine (FUdR) and the antifolate pemetrexed was analyzed using growth inhibition and clonogenic assays. Apoptosis was analyzed by flow cytometry. Significant variation in the quantity and cellular expression of dUTPase was observed, including clear evidence of overexpression in NSCLC cell line models and tumor specimens at the mRNA and protein level. RNA interference-mediated silencing of dUTPase significantly sensitized NSCLC cells to growth inhibition induced by FUdR and pemetrexed. This sensitization was accompanied by a significant expansion of intracellular dUTP pools and significant decreases in NSCLC cell viability evaluated by clonogenicity and apoptotic analyses. Together, these results strongly suggest that uracil misincorporation is a potent determinant of cytotoxicity to TS inhibition in NSCLC and that inhibition of dUTPase is a mechanism-based therapeutic approach to significantly enhance the efficacy of TS-targeted chemotherapeutic agents.

Prognostic significance of combining VEGFA, FLT1 and KDR mRNA expression in lung cancer

Angiogenesis is important in cancer progression. Promising results in clinical trials have indicated that targeting vascular epidermal growth factor (VEGF) signaling may prolong lung cancer patient survival. In particular, various studies have implicated VEGFA as a potential prognostic marker in lung cancer, although prognostication using the expression of VEGF receptors (VEGFRs), such as fms-related tyrosine kinase 1 (FLT1; also known as VEGFR1) and kinase insert domain receptor (KDR; also known as VEGFR2), has produced varied results in different lung cancer studies. The present study aimed to investigate the prognostic significance of these three factors, alone or in combination. mRNA expression data were extracted from four independent lung cancer cohorts totaling 583 patients, and the association between mRNA expression and survival was investigated by performing statistical analyses. When VEGFA, FLT1 and KDR expression were considered alone, only VEGFA demonstrated a significant association with patient survival consistently across all four datasets (P<0.05). Patients with a high expression of VEGFA and one of the two receptors were associated with significantly worse survival than patients expressing low levels of VEGFA and the particular receptor (P<0.05). Notably, patients with a high level expression of all three genes in their tumor specimens were associated with a significantly shorter survival time compared with patients exhibiting a low level expression of one, two or all three genes (P<0.05). The results indicate that a high level of VEGFA expression and its receptors may be required for cancer progression. Therefore, these three factors should be considered together as a prognostic indicator for lung cancer patients.

A comparison between accelerated hypofractionation and stereotactic ablative radiotherapy (SABR) for early-stage non-small cell lung cancer (NSCLC): Results of a propensity score-matched analysis

BACKGROUND AND PURPOSE: Stereotactic ablative radiotherapy (SABR) has become standard for inoperable early-stage non-small cell lung cancer (NSCLC). However, there is no randomized evidence demonstrating benefit over more fractionated radiotherapy. We compared accelerated hypofractionation (AH) and SABR using a propensity score-matched analysis.

MATERIALS AND METHODS: From 1997-2007, 119 patients (T1-3N0M0 NSCLC) were treated with AH (48-60Gy, 12-15 fractions). Prior to SABR, this represented our institutional standard. From 2008-2012, 192 patients (T1-3N0M0 NSCLC) were treated with SABR (48-52Gy, 4-5 fractions). A total of 114 patients (57 per cohort) were matched (1:1 ratio, caliper: 0.10) using propensity scores.

RESULTS: Median follow-up (range) for the AH cohort was 36.3 (2.5-109.1) months, while that for the SABR group was 32.5 (0.3-62.6)months. Three-year overall survival (OS) and local control (LC) rates were 49.5% vs. 72.4% [p=0.024; hazard ratio (HR): 2.33 (1.28, 4.23), p=0.006] and 71.9% vs. 89.3% [p=0.077; HR: 5.56 (1.53, 20.2), p=0.009], respectively. On multivariable analysis, tumour diameter and PET staging were predictive for OS, while the only predictive factor for LC was treatment cohort.

CONCLUSIONS: OS and LC were improved with SABR, although OS is more closely related to non-treatment factors. This represents one of the few studies comparing AH to SABR for early-stage lung cancer.

Protocol for the isotoxic intensity modulated radiotherapy (IMRT) in stage III non-small cell lung cancer (NSCLC): a feasibility study.

Introduction The majority of stage III patients with non-small cell lung cancer (NSCLC) are unsuitable for concurrent chemoradiotherapy, the non-surgical gold standard of care. As the alternative treatment options of sequential chemoradiotherapy and radiotherapy alone are associated with high local failure rates, various intensification strategies have been employed. There is evidence to suggest that altered fractionation using hyperfractionation, acceleration, dose escalation, and individualisation may be of benefit. The MAASTRO group have pioneered the concept of ‘isotoxic’ radiotherapy allowing for individualised dose escalation using hyperfractionated accelerated radiotherapy based on predefined normal tissue constraints. This study aims to evaluate whether delivering isotoxic radiotherapy using intensity modulated radiotherapy (IMRT) is achievable.

Methods and analysis Isotoxic IMRT is a multicentre feasibility study. From June 2014, a total of 35 patients from 7 UK centres, with a proven histological or cytological diagnosis of inoperable NSCLC, unsuitable for concurrent chemoradiotherapy will be recruited. A minimum of 2 cycles of induction chemotherapy is mandated before starting isotoxic radiotherapy. The dose of radiation will be increased until one or more of the organs at risk tolerance or the maximum dose of 79.2 Gy is reached. The primary end point is feasibility, with accrual rates, local control and overall survival our secondary end points. Patients will be followed up for 5 years.

Ethics and dissemination The study has received ethical approval (REC reference: 13/NW/0480) from the National Research Ethics Service (NRES) Committee North West—Greater Manchester South. The trial is conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (GCP). The trial results will be published in a peer-reviewed journal and presented internationally.

Trial registration number NCT01836692; Pre-results.

Metabolic signature of lung cancer: a metabolomic study of human tissues and biofluids

This thesis reports the application of metabolomics to human tissues and biofluids (blood plasma and urine) to unveil the metabolic signature of primary lung cancer. In Chapter 1, a brief introduction on lung cancer epidemiology and pathogenesis, together with a review of the main metabolic dysregulations known to be associated with cancer, is presented. The metabolomics approach is also described, addressing the analytical and statistical methods employed, as well as the current state of the art on its application to clinical lung cancer studies. Chapter 2 provides the experimental details of this work, in regard to the subjects enrolled, sample collection and analysis, and data processing. In Chapter 3, the metabolic characterization of intact lung tissues (from 56 patients) by proton High Resolution Magic Angle Spinning (HRMAS) Nuclear Magnetic Resonance (NMR) spectroscopy is described. After careful assessment of acquisition conditions and thorough spectral assignment (over 50 metabolites identified), the metabolic profiles of tumour and adjacent control tissues were compared through multivariate analysis. The two tissue classes could be discriminated with 97% accuracy, with 13 metabolites significantly accounting for this discrimination: glucose and acetate (depleted in tumours), together with lactate, alanine, glutamate, GSH, taurine, creatine, phosphocholine, glycerophosphocholine, phosphoethanolamine, uracil nucleotides and peptides (increased in tumours). Some of these variations corroborated typical features of cancer metabolism (e.g., upregulated glycolysis and glutaminolysis), while others suggested less known pathways (e.g., antioxidant protection, protein degradation) to play important roles. Another major and novel finding described in this chapter was the dependence of this metabolic signature on tumour histological subtype. While main alterations in adenocarcinomas (AdC) related to phospholipid and protein metabolisms, squamous cell carcinomas (SqCC) were found to have stronger glycolytic and glutaminolytic profiles, making it possible to build a valid classification model to discriminate these two subtypes. Chapter 4 reports the NMR metabolomic study of blood plasma from over 100 patients and near 100 healthy controls, the multivariate model built having afforded a classification rate of 87%. The two groups were found to differ significantly in the levels of lactate, pyruvate, acetoacetate, LDL+VLDL lipoproteins and glycoproteins (increased in patients), together with glutamine, histidine, valine, methanol, HDL lipoproteins and two unassigned compounds (decreased in patients). Interestingly, these variations were detected from initial disease stages and the magnitude of some of them depended on the histological type, although not allowing AdC vs. SqCC discrimination. Moreover, it is shown in this chapter that age mismatch between control and cancer groups could not be ruled out as a possible confounding factor, and exploratory external validation afforded a classification rate of 85%. The NMR profiling of urine from lung cancer patients and healthy controls is presented in Chapter 5. Compared to plasma, the classification model built with urinary profiles resulted in a superior classification rate (97%). After careful assessment of possible bias from gender, age and smoking habits, a set of 19 metabolites was proposed to be cancer-related (out of which 3 were unknowns and 6 were partially identified as N-acetylated metabolites). As for plasma, these variations were detected regardless of disease stage and showed some dependency on histological subtype, the AdC vs. SqCC model built showing modest predictive power. In addition, preliminary external validation of the urine-based classification model afforded 100% sensitivity and 90% specificity, which are exciting results in terms of potential for future clinical application. Chapter 6 describes the analysis of urine from a subset of patients by a different profiling technique, namely, Ultra-Performance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MS). Although the identification of discriminant metabolites was very limited, multivariate models showed high classification rate and predictive power, thus reinforcing the value of urine in the context of lung cancer diagnosis. Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the potential of integrated metabolomics of tissues and biofluids to improve current understanding of lung cancer altered metabolism and to reveal new marker profiles with diagnostic value.

Stereotactic body radiation therapy in stage I inoperable lung cancer: from palliative to curative options.

Surgery has historically been the standard of care for operable stage I non-small cell lung cancer (NSCLC). However, nearly one-quarter of patients with stage I NSCLC will not undergo surgery because of medical comorbidity or other factors. Stereotactic ablative radiotherapy (SABR) is the new standard of care for these patients. SABR offers high local tumour control rates rivalling the historical results of surgery and is generally well tolerated by patients with both peripheral and centrally located tumours. This article reviews the history of SABR for stage I NSCLC, summarises the currently available data on efficacy and toxicity, and describes some of the currently controversial aspects of this treatment.

Decision analysis of the effectiveness of lung cancer screening using autofluorescence bronchoscopy and computed tomography

Background: Lung cancer (LC) is the leading cause of cancer death in the developed world. Most cancers are associated with tobacco smoking. A primary hope for reducing lung cancer has been prevention of smoking and successful smoking cessation programs. To date, these programs have not been as successful as anticipated. Objective: The aim of the current study was to evaluate whether lung cancer screening combining low dose computed tomography with autofluorescence bronchoscopy (combined CT & AFB) is superior to CT or AFB screening alone in improving lung cancer specific survival. In addition, the extent of improvement and ideal conditions for combined CT & AFB screening were evaluated. Methods: We applied decision analysis and Monte Carlo simulation modeling using TreeAge Software to evaluate our study aims. Histology- and stage specific probabilities of lung cancer 5-year survival proportions were taken from Surveillance and Epidemiologic End Results (SEER) Registry data. Screeningassociated data was taken from the US NCI Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial (PLCO), National Lung Screening Trial (NLST), and US NCI Lung Screening Study (LSS), other relevant published data and expert opinion. Results: Decision Analysis - Combined CT and AFB was the best approach at Improving 5-year survival (Overall Expected Survival (OES) in the entire screened population was 0.9863) and in lung cancer patients only (Lung Cancer Specific Expected Survival (LOSES) was 0.3256). Combined screening was slightly better than CT screening alone (OES = 0.9859; LCSES = 0.2966), and substantially better than AFB screening alone (OES = 0.9842; LCSES = 0.2124), which was considerably better than no screening (OES = 0.9829; LCSES = 0.1445). Monte Carlo simulation modeling revealed that expected survival in the screened population and lung cancer patients is highest when screened using CT and combined CT and AFB. CT alone and combined screening was substantially better than AFB screening alone or no screening. For LCSES, combined CT and AFB screening is significantly better than CT alone (0.3126 vs. 0.2938, p< 0.0001). Conclusions: Overall, these analyses suggest that combined CT and AFB is slightly better than CT alone at improving lung cancer survival, and both approaches are substantially better than AFB screening alone or no screening.

Inhibition of human lung cancer cell proliferation and survival by wine

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.

Inhibition of human lung cancer cell proliferation and survival by wine

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events.

Associations Between Personal Cancer History and Lung Cancer Risk

The study aim was to investigate the relationship between factors related to personal cancer history and lung cancer risk as well as assess their predictive utility. Characteristics of interest included the number, anatomical site(s), and age of onset of previous cancer(s). Data from the Prostate, Lung, Colorectal and Ovarian Screening (PLCO) Cancer Screening Trial (N = 154,901) and National Lung Screening Trial (N = 53,452) were analysed. Logistic regression models were used to assess the relationships between each variable of interest and 6-year lung cancer risk. Predictive utility was assessed through changes in area-under-the-curve (AUC) after substitution into the PLCOall2014 lung cancer risk prediction model. Previous lung, uterine and oral cancers were strongly and significantly associated with elevated 6-year lung cancer risk after controlling for confounders. None of these refined measures of personal cancer history offered more predictive utility than the simple (yes/no) measure already included in the PLCOall2014 model.

Predicting the Risk of Lung Cancer in Never-smokers

Despite being considered a disease of smokers, approximately 10-15% of lung cancer cases occur in never-smokers. Lung cancer risk prediction models have demonstrated excellent ability to discriminate cases from non-cases, and have been shown to be more efficient at selecting individuals for future screening than current criteria. Existing models have primarily been developed in populations of smokers, thus there was a need to develop an accurate model in never-smokers. This study focused on developing and validating a model using never-smokers from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Cox regression analysis, with six-year follow-up, was used for model building. Predictors included: age, body mass index, education level, personal history of cancer, family history of lung cancer, previous chest X-ray, and secondhand smoke exposure. This model achieved fair discrimination (optimism corrected c-statistic = 0.6645) and good calibration. This represents an improvement on existing neversmoker models, but is not suitable for individual-level risk prediction.

Investigation of the Biological Effects of Rosemary (Rosmarinus Officinalis L.) Extract in Human Lung Cancer Cells

Cancer cells display enhanced growth rates and a resistance to apoptosis. Lung cancer accounts for the most cancer related deaths and non-small cell lung cancer (NSCLC) represents an aggressive form of lung cancer, accounting for almost 80% of all lung cancer cases. The phytochemical rosemary extract (RE) has been reported to have anticancer effects in vitro and in vivo however, limited evidence exists regarding the effects of RE and its polyphenolic constituents carnosic acid (CA) and rosmarinic acid (RA) in lung cancer. The present study shows RE, CA and RA inhibit lung cancer cell proliferation and survival in various NSCLC cell lines and that CA and RA interact synergistically to inhibit cell proliferation. Moreover RE, CA and RA are capable of altering activation and/or expression of Akt, ERK and AMPK, signaling molecules which regulate cell proliferation and survival. RE shows potential as an anticancer agent and should be further investigated.