Yes / It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival. Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reductionmediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a prerequisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator. / The authors would like to thank Cancer Research UK for the post-doctoral fellowship to H.F.Y.

No / Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit COX enzyme activity, a significant mechanism of action of NSAIDs. Inflammation is associated with increasing cancer incidence. Recent pre-clinical and clinical studies have shown that NSAID treatment could cause an anti-tumour effect in cancers. Such studies are lengthy and expensive. The present study, however, examined DNA damage in the Comet and micronucleus assays in peripheral blood lymphocytes of patients with respiratory diseases and healthy individuals using the nanoparticle (NP) and bulk versions of the NSAIDs, aspirin and ibuprofen. Lymphocytes are suitable surrogate cells for cancers and other disease states. DNA damage decreased in lymphocytes from healthy individuals, asthma, COPD and lung cancer patient groups after treatment with aspirin nano-suspension (ASP N) and ibuprofen nano-suspension (IBU N) compared to their bulk version (micro-suspension) in both assays. However, when ASP N was compared to untreated lymphocytes in all groups in the Comet assay, DNA damage significantly decreased in all groups, except the asthma group. When IBU N was compared to untreated lymphocytes, in healthy individuals and the lung cancer group, DNA damage decreased, but increased in asthma and COPD groups. Similarly, micronuclei (MNi) increased after ASP N and IBU N in the healthy individual and lung cancer groups, and decreased in asthma and COPD groups. Also shows that whilst there are basic similarities with different genetic endpoints in terms of nano and bulk forms, but highlights some differences between the disease states examined. Furthermore, lymphocyte responses after IBU N and ibuprofen bulk were investigated by patch-clamp experiments demonstrating that IBU N inhibited ion channel activity by 20%. This molecular epidemiology approach mirrors pre-clinical and clinical findings, and provides new information using nanoparticles.

Lung cancer is the leading cause of cancer-related deaths worldwide. One of the most common genetic aberrations in lung cancer patients is Kirsten rat sarcoma viral oncogene homolog (KRAS). The KRAS protein is a Ras superfamily GTPase that switches between an active GTP-bound form and an inactive GDP-bound form. The consequence of KRAS mutations results in constitutively active downstream pathways involved in uncontrolled cell proliferation and survival. Fortunately, there has been a recent development of KRASG12C inhibitors that directly target mutant KRAS, thereby arresting its proliferative effects. A recently FDA-approved KRASG12C inhibitor for the treatment of non-small cell lung cancer, Sotorasib (AMG 510), has been shown to produce insubstantial clinical response rates and a short duration of response. Similar to other targeted therapies, the limitations of this treatment are primarily due to the emergence of drug resistance. Drug resistance has been studied extensively regarding other anticancer treatments; however, the underlying molecular mechanisms remain poorly characterized. Our investigation begins by establishing and analyzing a subpopulation of cancer cells that evolve and mediate drug resistance, known as drug-tolerant persister cells (DTPCs), in KRASG12C mutant cells using AMG 510. First, we observed the reactivation of a pro-proliferative kinase, ERK, in AMG 510 DTPCs. Additionally, whole transcriptomics analysis, RT-qPCR, and immunofluorescent staining demonstrated significant upregulation of AQP4 in AMG 510 DTPCs compared to drug sensitive cells (DSCs). Aquaporin 4 (AQP4) is a water-selective transmembrane protein that regulates fluid homeostasis in many organ systems, including the lungs, and is involved in intracellular calcium signaling. We aim to explore the connections between AQP4, ERK, and calcium signaling in promoting drug tolerance to AMG 510. The insights gained from this research could lead to improved targeted therapies and clinical outcomes by identifying AQP4 as a resistance-driving biomarker.

Yes / Lung cancer remains a major cause of preventable death and early diagnosis is critical to improving survival chances. The chest X-ray (CXR) remains the most common initial investigation, but clinical pathways need to support timely diagnosis through, where necessary, escalation of abnormal findings to ensure priority reporting and early CT scan. This single-centre study included a retrospective evaluation of a rapid lung cancer CXR pathway in its first year of operation (May 2018-April 2019). The pathway was initially designed for primary care referrals but could also be used for any CXR demonstrating abnormal findings. A parallel cross-sectional survey of radiographers explored their understanding, adherence and concerns regarding their role in the pathway operation. Primary care referrals on the rapid diagnostic pathway were low (n = 51/21,980; 0.2%), with 11 (21.6%) requiring a CT scan. A further 333 primary care CXR were escalated by the examining radiographer, with 100 (30.0%) undergoing a CT scan. Overall, 64 of the CT scans (57.7%) were abnormal or demonstrated suspicious findings warranting further investigation. There were 39 confirmed primary lung carcinomas, most with advanced disease. Survey responses showed that most radiographers were familiar with the pathway but some expressed concerns regarding their responsibilities and limited knowledge of CXR pathologies. This baseline evaluation of the rapid lung cancer pathway demonstrated poor referral rates from primary care and identified the need for improved engagement. Radiographer escalation of abnormal findings is an effective adjunct but underlines the need for appropriate awareness, training, and ongoing support. Engagement of the multiprofessional team is critical in new pathway implementation. Rapid diagnostic pathways can enable early diagnosis and the radiographer has a key role to play in their success.

Disease mapping is used to identify high risk areas, inform resource allocation and generate hypotheses. The stroke and diabetes belts in the U.S. have encouraged public dialogue and spurred research. Lung cancer is the leading cause of U.S. cancer mortality, accounting for 158135 deaths in 2010 compared to 129180 from cerebrovascular disease and 68905 from diabetes mellitus. If one exists, defining a distinct pattern of high lung cancer mortality could increase public awareness of the disease and facilitate investigation of its determinants. To begin our inquiry, we generated a map and observed an area of high lung cancer mortality, primarily in the Southeast. However, variability in county rates, likely due to small populations, made determining patterns difficult. Spatial smoothing can clarify obscured patterns. We downloaded county lung cancer mortality rates, population sizes and death counts. Concurrent incidence and mortality rates for lung cancer were nearly equivalent, so mortality was used as a proxy for risk. After downloading county population centroids with latitudes and longitudes, we implemented a median-based, weighted, two-dimensional smoothing algorithm to enhance spatial patterns by borrowing strength from neighbor counties. The algorithm selected three proximate centroids, forming a “triple,” anchored by the centroid of the county to be smoothed. The parameter for nearest neighbor (NN) counties was set to NN=10, with the number of triples (NTR) for each county NTR=(2/3)*NN, producing seven collinear triples for each county with a center angle ≥135°. Median rates for the top and bottom 50% of neighbor counties were calculated and weighted by 1/SE, creating a “window,” whereby if the original rate was between the two medians, or if the county population was sufficiently large, it was not smoothed. If the original rate was outside the window, it was adjusted according to the corresponding neighbor median. Ten iterations of this process were conducted for each county. Smoothed rates were imported to ArcGIS and joined to a U.S. counties layer. Congruent counties in or near the Southeast with rates above 64 per 100,000 were defined as one class. We observed clustering of high lung cancer mortality, comprising 724 counties and forming an arc not evident in the unsmoothed data. This area, which we define as the lung cancer belt, included nearly all of Arkansas, Kentucky and Tennessee, and portions of 16 other states. Heavily affected regions include much of the Ohio Valley, Central Appalachia, the Tennessee Valley, the Ozarks, the Mississippi Delta and the northern Gulf Coast. Smoking, a modifiable behavior, causes the majority of lung cancer deaths, and is the single leading cause of mortality in the United States. Lung cancer mortality rates presented at the state level obscure differences within states. The lung cancer belt may provide a tool to identify areas in greatest need of resources. National survey data could be utilized to determine demographic, socioeconomic and behavioral differences between the lung cancer belt and the rest of the nation.

In most randomised controlled trials (RCTs), a large number of patients need to be followed over many years, for the clinical benefit of the drug to be accurately quantified (1). Using an early proxy, or a surrogate endpoint, in place of the direct endpoint of overall survival (OS) could theoretically shorten the duration of RCTs and minimise the exposure of patients to ineffective or toxic treatments (2, 3). This thesis examined the relationship between surrogate endpoints and OS in metastatic colorectal cancer (CRC), advanced non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). A review of the literature identified 144 RCTs in metastatic CRC, 189 in advanced NSCLC and 133 in MBC. The publications were generally of poor quality with incomplete reporting on many key variables, making comparisons between studies difficult. The introduction of the CONSORT statement was associated with improvements in the quality of reporting. For CRC (337 arms), NSCLC (429 arms) and MBC (290 arms) there were strong relationships between OS and progression free survival (PFS), time to progression (TTP), disease control rate (DCR), response rate (RR) and partial response (PR). Correlation was also demonstrated between OS and complete response (CR) in CRC and duration of response (DOR) in MBC. However, while strong relationships were found, the proportion of variance explained by the models was small. Prediction bands constructed to determine the surrogate threshold effect size indicated that large improvements in the surrogate endpoints were needed to predict overall survival gains. PFS and TTP showed the most promise as surrogates. The gain in PFS and TTP required to predict a significant gain in overall survival was between 1.2 and 7.0 months and 1.8 and 7.7 months respectively, depending on trial size and tumour type. DCR was a better potential predictor of OS than RR. The results of this study could be used to design future clinical trials with particular reference to the selection of surrogate endpoint and trial size.

In most randomised controlled trials (RCTs), a large number of patients need to be followed over many years, for the clinical benefit of the drug to be accurately quantified (1). Using an early proxy, or a surrogate endpoint, in place of the direct endpoint of overall survival (OS) could theoretically shorten the duration of RCTs and minimise the exposure of patients to ineffective or toxic treatments (2, 3). This thesis examined the relationship between surrogate endpoints and OS in metastatic colorectal cancer (CRC), advanced non-small cell lung cancer (NSCLC) and metastatic breast cancer (MBC). A review of the literature identified 144 RCTs in metastatic CRC, 189 in advanced NSCLC and 133 in MBC. The publications were generally of poor quality with incomplete reporting on many key variables, making comparisons between studies difficult. The introduction of the CONSORT statement was associated with improvements in the quality of reporting. For CRC (337 arms), NSCLC (429 arms) and MBC (290 arms) there were strong relationships between OS and progression free survival (PFS), time to progression (TTP), disease control rate (DCR), response rate (RR) and partial response (PR). Correlation was also demonstrated between OS and complete response (CR) in CRC and duration of response (DOR) in MBC. However, while strong relationships were found, the proportion of variance explained by the models was small. Prediction bands constructed to determine the surrogate threshold effect size indicated that large improvements in the surrogate endpoints were needed to predict overall survival gains. PFS and TTP showed the most promise as surrogates. The gain in PFS and TTP required to predict a significant gain in overall survival was between 1.2 and 7.0 months and 1.8 and 7.7 months respectively, depending on trial size and tumour type. DCR was a better potential predictor of OS than RR. The results of this study could be used to design future clinical trials with particular reference to the selection of surrogate endpoint and trial size.

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