5728 research outputs found
Sort by
Characterisation of WEE1 regulation at mitosis
No full textWee1 is a tyrosine kinase that plays a major role in regulating mitotic entry. Throughout G2 phase, it phosphorylates and inhibits Cdk1, the master mitotic regulator, thereby preventing mitotic entry. At the end of G2 phase Wee1 is inactivated, which causes the rapid activation of Cdk1 and irreversible entry into mitosis. It is important, therefore, that Wee1 activity is tightly regulated, to avoid premature mitotic entry in the face of incomplete DNA replication or cell stress including DNA damage. However, the regulation of Wee1 in human cells is poorly understood. My thesis aims to address this gap in knowledge by characterising Wee1 behaviour in RPE-1 cells. I use CRISPR-Cas9 technology to generate cell lines in which endogenous Wee1 is tagged with a fluorescent protein. Using live cell fluorescence microscopy, I show that Wee1 is neither degraded nor exported from the nucleus prior to mitosis, in contrast to previous reports. I also characterise the changes in phosphorylation of Wee1 across the cell cycle using mass spectrometry and identify a single phosphorylation site (S312) that, when mutated, causes a striking delay in G2/M progression. I use both live cell microscopy and immunofluorescence to show that expression of this Wee1 mutant causes defects in chromosome segregation and in the degradation of Cyclin A2 and B1 by APC/C during late mitosis. Finally, I carry out in vitro kinase assays to characterise the effect of this phosphosite mutation on the kinase activity of Wee1
Case-Case Genome-Wide Analyses Identify Subtype-Informative Variants That Confer Risk for Breast Cancer.
No full textBreast cancer includes several subtypes with distinct characteristic biological, pathologic, and clinical features. Elucidating subtype-specific genetic etiology could provide insights into the heterogeneity of breast cancer to facilitate the development of improved prevention and treatment approaches. In this study, we conducted pairwise case-case comparisons among five breast cancer subtypes by applying a case-case genome-wide association study (CC-GWAS) approach to summary statistics data of the Breast Cancer Association Consortium. The approach identified 13 statistically significant loci and eight suggestive loci, the majority of which were identified from comparisons between triple-negative breast cancer (TNBC) and luminal A breast cancer. Associations of lead variants in 12 loci remained statistically significant after accounting for previously reported breast cancer susceptibility variants, among which, two were genome-wide significant. Fine mapping implicated putative functional/causal variants and risk genes at several loci, e.g., 3q26.31/TNFSF10, 8q22.3/NACAP1/GRHL2, and 8q23.3/LINC00536/TRPS1, for TNBC as compared with luminal cancer. Functional investigation further identified rs16867605 at 8q22.3 as a SNP that modulates the enhancer activity of GRHL2. Subtype-informative polygenic risk scores (PRS) were derived, and patients with a high subtype-informative PRS had an up to two-fold increased risk of being diagnosed with TNBC instead of luminal cancers. The CC-GWAS PRS remained statistically significant after adjusting for TNBC PRS derived from traditional case-control GWAS in The Cancer Genome Atlas and the African Ancestry Breast Cancer Genetic Consortium. The CC-GWAS PRS was also associated with overall survival and disease-specific survival among patients with breast cancer. Overall, these findings have advanced our understanding of the genetic etiology of breast cancer subtypes, particularly for TNBC. Significance: The discovery of subtype-informative genetic risk variants for breast cancer advances our understanding of the etiologic heterogeneity of breast cancer, which could accelerate the identification of targets and personalized strategies for prevention and treatment
Bridging The Age Gap: observational cohort study of effects of chemotherapy and trastuzumab on recurrence, survival and quality of life in older women with early breast cancer.
No full textBACKGROUND: Chemotherapy improves outcomes for high risk early breast cancer (EBC) patients but is infrequently offered to older individuals. This study determined if there are fit older patients with high-risk disease who may benefit from chemotherapy. METHODS: A multicentre, prospective, observational study was performed to determine chemotherapy (±trastuzumab) usage and survival and quality-of-life outcomes in EBC patients aged ≥70 years. Propensity score-matching adjusted for variation in baseline age, fitness and tumour stage. RESULTS: Three thousands four hundred sixteen women were recruited from 56 UK centres between 2013 and 2018. Two thousands eight hundred eleven (82%) had surgery. 1520/2811 (54%) had high-risk EBC and 2059/2811 (73%) were fit. Chemotherapy was given to 306/1100 (27.8%) fit patients with high-risk EBC. Unmatched comparison of chemotherapy versus no chemotherapy demonstrated reduced metastatic recurrence risk in high-risk patients(hazard ratio [HR] 0.36 [95% CI 0.19-0.68]) and in 541 age, stage and fitness-matched patients(adjusted HR 0.43 [95% CI 0.20-0.92]) but no benefit to overall survival (OS) or breast cancer-specific survival (BCSS) in either group. Chemotherapy improved survival in women with oestrogen receptor (ER)-negative cancer (OS: HR 0.20 [95% CI 0.08-0.49];BCSS: HR 0.12 [95% CI 0.03-0.44]).Transient negative quality-of-life impacts were observed. CONCLUSIONS: Chemotherapy was associated with reduced risk of metastatic recurrence, but survival benefits were only seen in patients with ER-negative cancer. Quality-of-life impacts were significant but transient. TRIAL REGISTRATION: ISRCTN 46099296
Independent component analysis (ICA) applied to dynamic oxygen-enhanced MRI (OE-MRI) for robust functional lung imaging at 3 T.
No full textPURPOSE: Dynamic lung oxygen-enhanced MRI (OE-MRI) is challenging due to the presence of confounding signals and poor signal-to-noise ratio, particularly at 3 T. We have created a robust pipeline utilizing independent component analysis (ICA) to automatically extract the oxygen-induced signal change from confounding factors to improve the accuracy and sensitivity of lung OE-MRI. METHODS: Dynamic OE-MRI was performed on healthy participants using a dual-echo multi-slice spoiled gradient echo sequence at 3 T and cyclical gas delivery. ICA was applied to each echo within a thoracic mask. The ICA component relating to the oxygen-enhancement signal was automatically identified using correlation analysis. The oxygen-enhancement component was reconstructed, and the percentage signal enhancement (PSE) was calculated. The lung PSE of current smokers was compared with nonsmokers; scan-rescan repeatability, ICA pipeline repeatability, and reproducibility between two vendors were assessed. RESULTS: ICA successfully extracted a consistent oxygen-enhancement component for all participants. Lung tissue and oxygenated blood displayed the opposite oxygen-induced signal enhancements. A significant difference in PSE was observed between the lungs of current smokers and nonsmokers. The scan-rescan repeatability and the ICA pipeline repeatability were good. CONCLUSION: The developed pipeline demonstrated sensitivity to the signal enhancements of the lung tissue and oxygenated blood at 3 T. The difference in lung PSE between current smokers and nonsmokers indicates a likely sensitivity to lung function alterations that may be seen in mild pathology, supporting future use of our methods in patient studies
Respiratory motion modelling for MR-guided lung cancer radiotherapy: model development and geometric accuracy evaluation.
No full textObjective.Respiratory motion of lung tumours and adjacent structures is challenging for radiotherapy. Online MR-imaging cannot currently provide real-time volumetric information of the moving patient anatomy, therefore limiting precise dose delivery, delivered dose reconstruction, and downstream adaptation methods.Approach.We tailor a respiratory motion modelling framework towards an MR-Linac workflow to estimate the time-resolved 4D motion from real-time data. We develop a multi-slice acquisition scheme which acquires thick, overlapping 2D motion-slices in different locations and orientations, interleaved with 2D surrogate-slices from a fixed location. The framework fits a motion model directly to the input data without the need for sorting or binning to account for inter- and intra-cycle variation of the breathing motion. The framework alternates between model fitting and motion-compensated super-resolution image reconstruction to recover a high-quality motion-free image and a motion model. The fitted model can then estimate the 4D motion from 2D surrogate-slices. The framework is applied to four simulated anthropomorphic datasets and evaluated against known ground truth anatomy and motion. Clinical applicability is demonstrated by applying our framework to eight datasets acquired on an MR-Linac from four lung cancer patients.Main results.The framework accurately reconstructs high-quality motion-compensated 3D images with 2 mm3isotropic voxels. For the simulated case with the largest target motion, the motion model achieved a mean deformation field error of 1.13 mm. For the patient cases residual error registrations estimate the model error to be 1.07 mm (1.64 mm), 0.91 mm (1.32 mm), and 0.88 mm (1.33 mm) in superior-inferior, anterior-posterior, and left-right directions respectively for the building (application) data.Significance.The motion modelling framework estimates the patient motion with high accuracy and accurately reconstructs the anatomy. The image acquisition scheme can be flexibly integrated into an MR-Linac workflow whilst maintaining the capability of online motion-management strategies based on cine imaging such as target tracking and/or gating
Correlation analyses of radiographic progression-free survival with clinical and health-related quality of life outcomes in metastatic castration-resistant prostate cancer: Analysis of the phase 3 VISION trial.
No full textBACKGROUND: [177Lu]Lu-PSMA-617 (177Lu-PSMA-617) plus protocol-permitted standard of care (SOC) prolonged overall survival (OS) and radiographic progression-free survival (rPFS) versus SOC in patients with prostate-specific membrane antigen (PSMA)-positive metastatic castration-resistant prostate cancer (mCRPC) in the phase 3 VISION study, in addition to beneficial effects on symptomatic skeletal events (SSEs) and health-related quality of life (HRQOL). METHODS: Post hoc analyses used the full analysis set from the VISION study (N = 831) overall and by randomized treatment arm (177Lu-PSMA-617 plus SOC, n = 551; SOC, n = 280). Correlations were determined between OS and rPFS and between rPFS or OS and time to SSE or to worsening HRQOL (Functional Assessment of Cancer Therapy-Prostate [FACT-P] and 5-level EQ-5D [EQ-5D-5L]). Correlation analyses used an iterative multiple imputation copula-based approach (correlation coefficients [rho] of <0.3 were defined as weak, ≥0.3 and <0.5 as mild, ≥0.5 and <0.7 as moderate, and ≥0.7 as strong). RESULTS: In the overall population, rPFS correlated strongly with OS (rho, ≥0.7). Correlations between rPFS or OS and time to SSE without death were weak or mild. Time to worsening in the FACT-P total score and emotional and physical well-being domains correlated mildly or moderately with rPFS and moderately with OS. Correlation coefficients for time-to-worsening EQ-5D-5L scores were mild to moderate for both rPFS and OS. Correlation coefficients were similar between treatment arms. CONCLUSIONS: In this analysis of the VISION study, rPFS correlated strongly with OS but not with time to SSE or worsening HRQOL. These findings require further investigation
Development and Characterisation of Clinically Relevant Tissue Mimics for an Ultrasound Drug Delivery Phantom
Get PDFUltrasound (US) can be used for therapeutic applications, by exploiting the thermal and mechanical
effects induced in tissue. Knowledge of the acoustic, thermal and cavitation properties of tissue and
tissue mimicking materials (TMMs), which display the properties of different soft tissues, is essential
for experimental evaluation of therapeutic US. Furthermore, the use of TMMs to create a US drug
delivery phantom, will help in understanding the modalities and physical effects of cavitation.
The first two chapters of this thesis will review the current knowledge about therapeutic US,
characterisation techniques for relevant acoustic and thermal properties of tissues, and the need for
TMMs and phantoms, and potential candidates for these. The thesis will then focus on TMM
development, and the methodologies used here for acoustic, thermal and cavitation property
characterisation of tissue and TMMs, moving on to the results and their discussion. The sixth
chapter will focus on the design, development and set up of a US drug delivery phantom. The final
chapter will summarise the overall work and give conclusions, and suggest potential future wor
Development of Real-Time Motion-Including Dose Reconstruction on a Unity MR-Linac system
No full textThe first part of my thesis is concerned with estimating the motion of organs and other critical anatomical structures during radiotherapy treatments. I use 2D cine images acquired during MR-Linac treatments and compare different computer vision techniques to obtain motion vectors from these.Then I calculate the radiation dose received by the patient in “real time” (as they are being treated). I use a clinically validated fast Monte Carlo dose calculation algorithm, and I iteratively compute beam doses during radiation delivery and accumulate the dose cubes on a 3D representation of the patient. In each iteration I include the current state of the patient anatomy determined in the first part. Thus I am able to compute the radiation received by moving anatomy whilst the treatment is underway. Finally, I investigate the clinical relevance of this technique. There are two avenues that I explore: the first is to facilitate the use of real-time intelligent correction measures, such as gating and tumour tracking. The second is to create prediction models to determine whether the radiation dose delivered to the target structure and organs at risk will be suboptimal due to the presence of motion. If suboptimal dosimetry is detected, it could be corrected for by various strategies, thus improving patient outcomes
Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.
No full textThe phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies
Melanoma cell morphogenesis in 3D environments
No full textDuring metastasis cancer cells invade tissues with diverse rigidities through dynamic changes in their cell shape. Most of our understanding of cancer cells shape determination comes from 2D studies on extremely stiff plastic substrates. As such, there is little insight into cancer cell morphogenesis on physiologically relevant matrices, and in 3D space. Rho GTP Exchange Factors (RhoGEFs) and Rho GTPase Activating Proteins (RhoGAPs) are large and diverse families of molecules that play a role in eukaryotic shape determination. Though most remain uncharacterized, RhoGEFs and RhoGAPs are ideal candidates for proteins capable of coupling melanoma morphogenesis to variations in matrix rigidity and geometry. To identify RhoGEFs and RhoGAPs that play a role in cell shape determination on soft matrices I performed genetic screens of cells cultured on soft collagen hydrogels in tandem with quantitative morphological analysis. From the screens I found that ARHGEF9/collybistin is essential for cell shape determination on both soft and stiff matrices, and in cells embedded in 3D. I show that ARHGEF9 promotes the formation of actin rich filopodia, which serve to establish and stabilize focal adhesions. Depletion of ARHGEF9 results in loss of tension at adhesions, decreased cell-wide contractility, and the inability to stabilize protrusions. Moreover, ARHGEF9 is required for melanoma cells to invade 3D matrices. The role of ARHGEF9 in shape determination and focal adhesion assembly is dependent on CDC42. I propose that ARHGEF9 may confer melanoma cells with the ability to explore and adhere to extracellular matrices. In addition, melanoma metastasis in 3D matrices is like how endothelial or neuronal cells migrate through the body during development.Abnormal cell cycle progression is one of the hallmarks of cancer cells (Hanahan and Weinberg, 2011). In this study, I show that ARHGEF9 influences the cell cycle progression. Melanoma cells depleted of ARHGEF9 have a slower growth rate and significantly lower levels of pRB compared to control cells. Thus, ARHGEF9 depleted cells enter the cell cycle with defects at the initial stage which influences their proliferation rate. My data demonstrates that ARHGEF9 can potentially be a target for slowing the uncontrolled growth rate of melanoma cells which results in tumorigenesis