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Bimodal Electric Tissue Ablation (BETA) combines direct current with conventional RFA with a resultant increase in the ablation zone size, and an effect on the needle that makes it much harder for tissue to adhere to it. To explore the effects of BETA, ex vivo and in vivo studies were conducted. BETA was found to create a larger ablation zones compared to conventional RFA (p<0.0001). The hypothesis for this observation is the net movement of water through tissue, a process termed electroosmosis. Analysis of samples treated with BETA and conventional RFA showed a significantly higher hydration percentage following ablation with BETA (p<0.0001). Temperature distribution studies demonstrated cytocidal temperatures at 5, 10, 15 and 20mm from the electrode following BETA (p<0.0001). In order to assess the effects of BETA in vivo, large animals studies were conducted. Twelve pigs underwent four 'open' conventional RFA cycles and four BETA cycles. These studies showed significantly larger ablation zones following BETA compared to conventional RFA (p<0.0001), with no local complications observed. The inflammatory response to BETA was investigated; pig Major Acute Phase Protein, Serum Amyloid A, Haptoglobin and C Reactive Protein assays were analysed pre and post ablation. The proteins peaked at 48 and 72 hours and all returned to normal levels at termination. The outcome of this research demonstrates BETA to produce significantly larger ablation zones due to increased hydration of the ablated tissue, with superior temperature distribution and comparable systemic and clinical effects in animal models.

In this thesis, we develop image analysis techniques, applied and tested in a clinical environment, to support the management of patients with (metastatic) liver cancer The incidence of this cancer is rising and represents approximately 10% of cancer cases in men and women. Image analysis of the liver is difficult, in part because it is the only organ mixing arterial and (portal) venous blood, and in part because of the large excursion it is undergoing during respiration.

Hepatocellular carcinoma (HCC) is the sixth most common cancer and third most common cause of cancer-related death worldwide. Inadequate screening tests largely account for presentation of advanced tumours, poor prognosis and high mortality rates. Major risk factors for the development of HCC include Hepatitis B and C Virus infections and alcoholism, which lead to irreversible liver cirrhosis. Early detection of HCC amongst high-risk groups is paramount in improving prognosis, through enabling curative treatment options to be administered prior to manifestation of advanced and metastatic disease. An elicited humoral immune response, in the form of IgG autoantibodies raised to tumour-associated antigens, has been evidenced in the sera of cancer patients. Autoantibody measurements, using a minimally invasive blood test, could serve as an aid to early diagnosis by providing a window on early carcinogenesis months to years before the tumour bulk becomes otherwise clinically detectable. Autoantibody detection has previously been described in the HCC setting. Small numbers of antigens and use of sub-optimal control sera render published studies open to scrutiny. This thesis documents the research carried out in order to characterise the previously documented autoantibody response in the sera of patients with HCC and underlying liver disease. Tumour-associated antigens were produced using high-throughput recombinant technology and screened against sera from patients with HCC, liver disease and matched healthy controls. Varying autoantibody specificities and sensitivities were observed to the individual tumour-associated antigens investigated. A panel of 21 antigens achieved a specificity of 92% and sensitivity of 45% for the detection of HCC (by testing 96 cancers and 96 matched healthy controls). This same panel identified 21 % of 169 high-risk controls as having elevated autoantibody levels. This highlights the need for further investigation in order to determine whether such specificity (79%) would be deemed acceptable in a clinical setting for the application of an autoantibody test to aid in earlier cancer diagnosis. A reproducible panel of II antigens achieved a specificity of 91 % and sensitivity of 41 % for the detection of HCC, suggesting the potential held by optimised antigen production and careful panel selection. Changing autoantibody profiles in the sera of patients with stable cirrhosis and in the pre- and post-diagnostic sera of patients with HCC suggests a potential role for autoantibodies and their detection in the surveillance, diagnosis and prediction of recurrence in the HCC setting. Results are comparable to current gold standards in HCC (Ultrasonography) and to similar tests in other cancers (Early-CDT®-Lung). This simple blood test has the potential to offer a competitive advantage over currently available tools for the early identification of HCC amongst patients routinely monitored for the progression of their underlying liver disease.

Primary liver cancer, which consists of predominantly hepatocellular carcinoma (HCC), is the fifth most common cancer worldwide and the third most common cause of cancer mortality. The majority of patients are diagnosed with advanced disease when only palliative therapies are an option. HCC are notoriously resistant to chemotherapies. Several key DNA damage signalling and repair genes were significantly up-regulated in HCC. Most marked was the up-regulation in line with progression, of the PRKDC gene, the product of which is DNA-PKcs, the key enzyme which orchestrates the non-homologous end joining pathway of DNA double strand break repair. Increased PRKDC expression was also observed in non-alcoholic fatty liver disease, increasing with degree of inflammation, implicating DNA-PK in the pathogenesis of chronic liver disease, HCC, and possibly chemotherapy resistance. Inhibition of DNA-PK by NU7441 caused substantial retardation of early, NHEJ-mediated, DSB signalling and repair, and increased dependence on homologous recombination in HCC cell lines. NU7441 also significantly potentiated ionizing radiation and doxorubicin induced cytotoxicity, causing a dramatic decrease in cell growth and survival. Furthermore, whilst ATM inhibition by KU55933 or DNA-PK inhibition by NU7441 alone had no effect on cell survival, the combination was cytotoxic to all HCC cell lines used. DNA-PK protein levels and activity were substantially higher in HCC tumour tissues than non-tumour liver tissue, and further increased with tumour grade. Increased tumour DNAPK levels were associated with reduced time to progression and poorer survival in patients following transarterial chemoembolization with DNA-damaging agent, doxorubicin. Also, DNA-PK activity was an independent predictor of survival in a cohort of 44 HCC patients. These data suggest patients with high DNA-PK protein levels and/or activity may benefit from the inclusion of a DNA-PK inhibitor either alone, in combination with a DNA-damaging agent such as doxorubicin, or in combination with an ATM inhibitor.

Liver surgical resection is one of the most frequently used curative therapies. However, resectability is problematic. There is a need for a computer-assisted surgical planning and simulation system which can accurately and efficiently simulate the liver, vessels and tumours in actual patients. The present project describes the development of these core segmentation and deformable modelling techniques. For precise detection of irregularly shaped areas with indistinct boundaries, the segmentation incorporated active contours - gradient vector flow (GVF) snakes and level sets. To improve efficiency, a chessboard distance transform was used to replace part of the GVF effort. To automatically initialize the liver volume detection process, a rotating template was introduced to locate the starting slice. For shape maintenance during the segmentation process, a simplified object shape learning step was introduced to avoid occasional significant errors. Skeletonization with fuzzy connectedness was used for vessel segmentation. To achieve real-time interactivity, the deformation regime of this system was based on a single-organ mass-spring system (MSS), which introduced an on-the-fly local mesh refinement to raise the deformation accuracy and the mesh control quality. This method was now extended to a multiple soft-tissue constraint system, by supplementing it with an adaptive constraint mesh generation. A mesh quality measure was tailored based on a wide comparison of classic measures. Adjustable feature and parameter settings were thus provided, to make tissues of interest distinct from adjacent structures, keeping the mesh suitable for on-line topological transformation and deformation. More than 20 actual patient CT and 2 magnetic resonance imaging (MRI) liver datasets were tested to evaluate the performance of the segmentation method. Instrument manipulations of probing, grasping, and simple cutting were successfully simulated on deformable constraint liver tissue models. This project was implemented in conjunction with the Division of Surgery, Hammersmith Hospital, London; the preliminary reality effect was judged satisfactory by the consultant hepatic surgeon.

In the last 20 years laparoscopic liver resection has been increasingly practised. However its role in the treatment of liver cancer remains under scrutiny. I performed a pilot study at a specialist HPB unit assessing the results of the laparoscopic resections of one surgeon and comparing them to the results of matched cases on whom he had performed open resection. The resection technique was radiofrequency assisted resection pioneered in this unit. I also analysed the resected tissue to investigate any differential effect on cell characteristics of the 2 operative techniques. Operative time was significantly longer in laparoscopic cases and time to recurrence of R0 resections significantly shorter. Resected tissue demonstrated significantly higher levels of the hypoxia inducible factor-2 and CD10, a recognised poor prognostic marker in primary colorectal tumours. I hypothesised that livers resected laparoscopically are under a relative hypoxia because of the increased intraabdominal pressure associated with the pneumoperitoneum and tumours cells therefore have a positive selection advantage. In the setting of longer resection times this may compromise the oncological result of the surgery causing earlier recurrence. Using a established model of HIF activation, I showed that poor prognostic marker CD10 may be a function of hypoxia inducible regulation. Certainly I was able to replicate data from cervical squamous epithelia demonstrating that both in benign, dysplastic and malignant tissue, HIF expression corresponded to a reduced cell E-cadherin expression that may allow a more malignant potential. I also analysed the effect of RF ablation on circulating tumour cells in palliative irresectable cancers and in the context of both open and laparoscopic liver resection. This showed only a transient rise in both resectional techniques, (open and lap) that would unlikely count for the differential oncological outcome previously demonstrated in the pilot study. In keeping with current international opinion, further work is required to verify the role of laparoscopic liver resection in liver cancer.

Hepatocellular carcinoma (HCC) is a devastating malignancy originating from hepatocytes. There is an urgent need for novel therapeutic approaches. Currently explored gene therapy systems have not yet achieved significant survival benefits. The aim of this thesis was the development and evaluation of novel genetic approaches to this malignancy.

Clonally derived liver tumours and an ovarian cyst developed in mice following EIAV and FIV delivery in utero. LAM PCR and 454 sequencing was used to retrieve proviral insertion sites. TaqMan analysis revealed gene expression changes in lentiviral infected tumours. STRING and IPA networks identified links between genes flanking the lentivirus provirus and oncogenic pathways supporting the role of insertional mutagenesis in Hepatocellular Carcinoma (HCC). Global methylation analysis demonstrated increased relative methylation levels in lentivirus (EIAV, FIV, and HIV) infected normal and tumour samples. This provided strong evidence for host defence against lentivirus infection by epigenetic means. Microarray data showed altered expression of Dnmt1 and Dnmt3b and TaqMan analysis revealed specific changes in Dnmts levels when compared to uninfected liver. The evidence found for involvement of DNA methylation associated with lentivirus infection and possibly tumour development required that this study be repeated in vitro. DNA methylation was investigated at early time points after lentivirus and retrovirus infection in HepG2 cells. Results revealed sharp increases in global methylation and Dnmt levels at 24 and 30hrs post infection. E2F targets play a key role in the regulation of gene expression and aberrations result in the development of cancer. Of the 94 E2F target genes analysed 77.7% were involved in DNA damage and repair mechanisms, 21.3% were known oncogenes or shown to exert oncogenic activity and 80.9% were categorised as HCC target genes. The fact that all lentiviral/retroviral vectors used in this study were found to cause changes in methylation and gene expression in vivo and in vitro suggests that these vectors, at least in the mouse, are genotoxic. Findings here support the use of the fetal animal model to identify vector genotoxicity and the mechanisms of lentiviral vector-induced tumorigenesis. This model may be a valuable tool to evaluate the safety of lentiviruses for gene therapy.