Three dimensional study to quantify the relationship between facial hard and soft tissue movement as a result of orthognathic surgery

Introduction Prediction of soft tissue changes following orthognathic surgery has been frequently attempted in the past decades. It has gradually progressed from the classic “cut and paste” of photographs to the computer assisted 2D surgical prediction planning; and finally, comprehensive 3D surgical planning was introduced to help surgeons and patients to decide on the magnitude and direction of surgical movements as well as the type of surgery to be considered for the correction of facial dysmorphology. A wealth of experience was gained and numerous published literature is available which has augmented the knowledge of facial soft tissue behaviour and helped to improve the ability to closely simulate facial changes following orthognathic surgery. This was particularly noticed following the introduction of the three dimensional imaging into the medical research and clinical applications. Several approaches have been considered to mathematically predict soft tissue changes in three dimensions, following orthognathic surgery. The most common are the Finite element model and Mass tensor Model. These were developed into software packages which are currently used in clinical practice. In general, these methods produce an acceptable level of prediction accuracy of soft tissue changes following orthognathic surgery. Studies, however, have shown a limited prediction accuracy at specific regions of the face, in particular the areas around the lips. Aims The aim of this project is to conduct a comprehensive assessment of hard and soft tissue changes following orthognathic surgery and introduce a new method for prediction of facial soft tissue changes.   Methodology The study was carried out on the pre- and post-operative CBCT images of 100 patients who received their orthognathic surgery treatment at Glasgow dental hospital and school, Glasgow, UK. Three groups of patients were included in the analysis; patients who underwent Le Fort I maxillary advancement surgery; bilateral sagittal split mandibular advancement surgery or bimaxillary advancement surgery. A generic facial mesh was used to standardise the information obtained from individual patient’s facial image and Principal component analysis (PCA) was applied to interpolate the correlations between the skeletal surgical displacement and the resultant soft tissue changes. The identified relationship between hard tissue and soft tissue was then applied on a new set of preoperative 3D facial images and the predicted results were compared to the actual surgical changes measured from their post-operative 3D facial images. A set of validation studies was conducted. To include: • Comparison between voxel based registration and surface registration to analyse changes following orthognathic surgery. The results showed there was no statistically significant difference between the two methods. Voxel based registration, however, showed more reliability as it preserved the link between the soft tissue and skeletal structures of the face during the image registration process. Accordingly, voxel based registration was the method of choice for superimposition of the pre- and post-operative images. The result of this study was published in a refereed journal. • Direct DICOM slice landmarking; a novel technique to quantify the direction and magnitude of skeletal surgical movements. This method represents a new approach to quantify maxillary and mandibular surgical displacement in three dimensions. The technique includes measuring the distance of corresponding landmarks digitized directly on DICOM image slices in relation to three dimensional reference planes. The accuracy of the measurements was assessed against a set of “gold standard” measurements extracted from simulated model surgery. The results confirmed the accuracy of the method within 0.34mm. Therefore, the method was applied in this study. The results of this validation were published in a peer refereed journal. • The use of a generic mesh to assess soft tissue changes using stereophotogrammetry. The generic facial mesh played a major role in the soft tissue dense correspondence analysis. The conformed generic mesh represented the geometrical information of the individual’s facial mesh on which it was conformed (elastically deformed). Therefore, the accuracy of generic mesh conformation is essential to guarantee an accurate replica of the individual facial characteristics. The results showed an acceptable overall mean error of the conformation of generic mesh 1 mm. The results of this study were accepted for publication in peer refereed scientific journal. Skeletal tissue analysis was performed using the validated “Direct DICOM slices landmarking method” while soft tissue analysis was performed using Dense correspondence analysis. The analysis of soft tissue was novel and produced a comprehensive description of facial changes in response to orthognathic surgery. The results were accepted for publication in a refereed scientific Journal. The main soft tissue changes associated with Le Fort I were advancement at the midface region combined with widening of the paranasal, upper lip and nostrils. Minor changes were noticed at the tip of the nose and oral commissures. The main soft tissue changes associated with mandibular advancement surgery were advancement and downward displacement of the chin and lower lip regions, limited widening of the lower lip and slight reversion of the lower lip vermilion combined with minimal backward displacement of the upper lip were recorded. Minimal changes were observed on the oral commissures. The main soft tissue changes associated with bimaxillary advancement surgery were generalized advancement of the middle and lower thirds of the face combined with widening of the paranasal, upper lip and nostrils regions. In Le Fort I cases, the correlation between the changes of the facial soft tissue and the skeletal surgical movements was assessed using PCA. A statistical method known as ’Leave one out cross validation’ was applied on the 30 cases which had Le Fort I osteotomy surgical procedure to effectively utilize the data for the prediction algorithm. The prediction accuracy of soft tissue changes showed a mean error ranging between (0.0006mm±0.582) at the nose region to (-0.0316mm±2.1996) at the various facial regions.

An investigation of the clinical utility of preoperative cardiopulmonary exercise testing in patients undergoing major pancreatic surgery

Pancreaticoduodenectomy with or without adjuvant chemotherapy remains the only modality of possible cure in patients with cancer involving the head of the pancreas and the periampullary region. While mortality rates after pancreaticoduodenectomy have improved considerably over the course of the last century, morbidity remains high. Patient selection is of paramount importance in ensuring that major surgery is offered to individuals who will most benefit from a pancreaticoduodenectomy. Moreover, identifying preoperative risk factors provides potential targets for prehabilitation and optimisation of the patient's physiology before undertaking surgery. In addition to this, early identification of patients who are likely to develop postoperative complications allows for better allocation of critical care resources and more aggressive management high risk patients. Cardiopulmonary exercise testing is becoming an increasingly popular tool in the preoperative risk assessment of the surgical patient. However, very little work has been done to investigate the role of cardiopulmonary exercise testing in predicting complications after pancreaticoduodenectomy. The impact of jaundice, systemic inflammation and other preoperative clinicopathological characteristics on cardiopulmonary exercise physiology has not been studied in detail before in this cohort of patients. The overall aim of the thesis was to examine the relationships between preoperative clinico-pathological characteristics including cardiopulmonary exercise physiology, obstructive jaundice, body composition and systemic inflammation and complications and the post-surgical systemic inflammatory response in patients undergoing pancreaticoduodenectomy. Chapter 1 reviews the existing literature on preoperative cardiopulmonary exercise testing, the impact of obstructive jaundice, perioperative systemic inflammation and the importance of body composition in determining outcomes in patients undergoing major surgery with particular reference to pancreatic surgery. Chapter 2 reports on the role of cardiopulmonary exercise testing in predicting postoperative complications after pancreaticoduodenectomy. The results demonstrate that patients with V˙O2AT less than 10 ml/kg/min are more likely to develop a postoperative pancreatic fistula, stay longer in hospital and less likely to receive adjuvant therapy. These results emphasise the importance of aerobic fitness to recover from the operative stress of major surgery without significant morbidity. Cardiopulmonary exercise testing may prove useful in selecting patients for intensive prehabilitation programmes as well as for other optimisation measures to prepare them for major surgery. Chapter 3 evaluates the relationship between cardiopulmonary exercise physiology and other clinicopathological characteristics of the patient. A detailed analysis of cardiopulmonary exercise test parameters in jaundiced versus non-jaundiced patients demonstrates that obstructive jaundice does not impair cardiopulmonary exercise physiology. This further supports emerging evidence in contemporary literature that jaundiced patients can proceed directly to surgery without preoperative biliary drainage. The results of this study also show an interesting inverse relationship between body mass index and anaerobic threshold which is analysed in more detail in Chapter 4. Chapter 4 examines the relationship between preoperative cardiopulmonary exercise physiology and body composition in depth. All parameters measured at cardiopulmonary exercise test are compared against body composition and body mass index. The results of this chapter report that the current method of reporting V˙O2, both at peak exercise and anaerobic threshold, is biased against obese subjects and advises caution in the interpretation of cardiopulmonary exercise test results in patients with a high BMI. This is particularly important as current evidence in literature suggests that postoperative outcomes in obese subjects are comparable to non-obese subjects while cardiopulmonary exercise test results are also abnormally low in this very same cohort of patients. Chapter 5 analyses the relationship between preoperative clinico-pathological characteristics including systemic inflammation and the magnitude of the postoperative systemic inflammatory response. Obstructive jaundice appears to have an immunosuppressive effect while elevated preoperative CRP and hypoalbuminemia appear to have opposite effects with hypoalbuminemia resulting in a lower response while elevated CRP in the absence of hypoalbuminemia resulted in a greater postoperative systemic inflammatory response. Chapter 6 evaluates the role of the early postoperative systemic inflammatory response in predicting complications after pancreaticoduodenectomy and aims to establish clinically relevant thresholds for C-Reactive Protein for the prediction of complications. The results of this chapter demonstrate that CRP levels as early as the second postoperative day are associated with complications. While post-operative CRP was useful in the prediction of infective complications, this was the case only in patients who did not develop a post-operative pancreatic fistula. The predictive ability of inflammatory markers for infectious complications was blunted in patients with a pancreatic fistula. Chapter 7 summarises the findings of this thesis, their place in current literature and future directions. The results of this thesis add to the current knowledge regarding the complex pathophysiological abnormalities in patients undergoing pancreaticoduodenectomy, with specific emphasis on the interaction between cardiopulmonary exercise physiology, obstructive jaundice, systemic inflammation and postoperative outcomes. The work presented in this thesis lays the foundations for further studies aimed at improving outcomes after pancreaticoduodenectomy through the development of individualised, goal-directed therapies that are initiated well before this morbid yet necessary operation is performed.

Effect of remote ischaemic preconditioning in cardiac dysfunction and end-organ injury following cardiac surgery with cardiopulmonary bypass in children: A translational approach investigating clinical outcome and myocardial molecular biology

Congenital heart disease (CHD) is the most common birth defect, causing an important rate of morbidity and mortality. Treatment of CHD requires surgical correction in a significant percentage of cases which exposes patients to cardiac and end organ injury. Cardiac surgical procedures often require the utilisation of cardiopulmonary bypass (CPB), a system that replaces heart and lungs function by diverting circulation into an external circuit. The use of CPB can initiate potent inflammatory responses, in addition a proportion of procedures require a period of aortic cross clamp during which the heart is rendered ischaemic and is exposed to injury. High O2 concentrations are used during cardiac procedures and when circulation is re-established to the heart which had adjusted metabolically to ischaemia, further injury is caused in a process known as ischaemic reperfusion injury (IRI). Several strategies are in place in order to protect the heart during surgery, however injury is still caused, having detrimental effects in patients at short and long term. Remote ischaemic preconditioning (RIPC) is a technique proposed as a potential cardioprotective measure. It consists of exposing a remote tissue bed to brief episodes of ischaemia prior to surgery in order to activate protective pathways that would act during CPB, ischaemia and reperfusion. This study aimed to assess RIPC in paediatric patients requiring CHD surgical correction with a translational approach, integrating clinical outcome, marker analysis, cardiac function parameters and molecular mechanisms within the cardiac tissue. A prospective, single blinded, randomized, controlled trial was conducted applying a RIPC protocol to randomised patients through episodes of limb ischaemia on the day before surgery which was repeated right before the surgery started, after anaesthesia induction. Blood samples were obtained before surgery and at three post-operative time points from venous lines, additional pre and post-bypass blood samples were obtained from the right atrium. Myocardial tissue was resected during the ischaemic period of surgery. Echocardiographic images were obtained before the surgery started after anaesthetic induction and the day after surgery, images were stored for later off line analysis. PICU surveillance data was collected including ventilation parameters, inotrope use, standard laboratory analysis and six hourly blood gas analysis. Pre and post-operative quantitation of markers in blood specimens included cardiac troponin I (cTnI) and B-type natriuretic peptide (BNP), inflammatory mediators including interleukins IL-6, IL-8, IL-10, tumour necrosis factor (TNF-α), and the adhesion molecules ICAM-1 and VCAM-1; the renal marker Cystatin C and the cardiovascular markers asymmetric dymethylarginine (ADMA) and symmetric dymethylarginine (SDMA). Nitric oxide (NO) metabolites and cyclic guanosine monophosphate (cGMP) were measured before and after bypass. Myocardial tissue was processed at baseline and after incubation at hyperoxic concentration during four hours in order to mimic surgical conditions. Expression of genes involved in IRI and RIPC pathways was analysed including heat shock proteins (HSPs), toll like receptors (TLRs), transcription factors nuclear factor κ-B (NF- κ-B) and hypoxia inducible factor 1 (HIF-1). The participation of hydrogen sulfide enzymatic genes, apelin and its receptor were explored. There was no significant difference according to group allocation in any of the echocardiographic parameters. There was a tendency for higher cTnI values and inotropic score in control patients post-operatively, however this was not statistically significant. BNP presented no significant difference according to group allocation. Inflammatory parameters tended to be higher in the control group, however only TNF- α was significantly higher. There was no difference in levels of Cystatin C, NO metabolites, cGMP, ADMA or SDMA. RIPC patients required shorter PICU stay, all other clinical and laboratory analysis presented no difference related to the intervention. Gene expression analysis revealed interesting patterns before and after incubation. HSP-60 presented a lower expression at baseline in tissue corresponding to RIPC patients, no other differences were found. This study provided with valuable descriptive information on previously known and newly explored parameters in the study population. Demographic characteristics and the presence of cyanosis before surgery influenced patterns of activity in several parameters, numerous indicators were linked to the degree of injury suffered by the myocardium. RIPC did not reduce markers of cardiac injury or improved echocardiographic parameters and it did not have an effect on end organ function; some effects were seen in inflammatory responses and gene expression analysis. Nevertheless, an important clinical outcome indicator, PICU length of stay was reduced suggesting benefit from the intervention. Larger studies with more statistical power could determine if the tendency of lower injury and inflammatory markers linked to RIPC is real. The present results mostly support findings of larger multicentre trials which have reported no cardiac benefit from RIPC in paediatric cardiac surgery.

Advanced scrub practitioner toolkit

Toolkit for operating department staff and operating departments for role development NAASP (National Association Of Assistants In Surgical Practice) advanced practitioner toolkit provides outline strategies and a framework within the advanced scrub practitioner (ASP) role can be developed. this document defines the range of knowledge and skills required and sets the standards of practice for the ASP.

Effectiveness of diode laser trans-scleral cyclophotocoagulation in patients following silicone oil-induced ocular hypertension in Chinese eyes.

We evaluated the effectiveness of diode laser trans-scleral cyclophotocoagulation (TSCPC) on intraocular pressure (IOP) in nine patients having raised IOP following use of silicone oil (SO) for retinal detachment (RD) surgery in a retrospective observational case series. Diode laser TSCPC was applied at a power setting of 1.75 to 2.5 watts, for two sec with a maximum of 30 applications. The patients were followed up for 40 to 312 weeks. The mean pre-laser IOP was 32.06 mm Hg (SD 7.32). The mean post-laser IOP at one month, three months and six months was 17.89 mm Hg (SD 8.23), 21.89 mm Hg (SD 8.16) and 21.67 mm Hg (SD 7.55) respectively. The final IOP (at the last follow-up) was 19.56 mm Hg (SD 7.85) (P=0.021). Seven of them had undergone SO removal. In our observation, effectiveness of TSCPC in long-term control of SO-induced ocular hypertension was limited as compared to short-term control of IOP.

Factores asociados a recuperación visual deficiente después de cirugía exitosa para desprendimiento de retina regmatógeno

Introducción: Varias características pueden afectar el pronóstico visual después de resolver quirúrgicamente el desprendimiento de retina. Existen características no observables por el ojo humano por si solo pero si por tomografía óptica coherente que se relacionan con la recuperación visual. Objetivo: Describir las características clínicas y topográfica en los periodos pre y postquirúrgico de ojos que ha sufrido DR regmatógeno con compromiso macular y su relación con la calidad de recuperación visual después de una cirugía considerada exitosa desde el punto de vista anatómico. Materiales y métodos: Estudio descriptivo en el que se comparan algunas características en tres periodos perioeperatorios, uno antes y dos después de cirugía (3 y 6 meses) de 24 ojos con DRregmatógeno y compromiso macular intervenidos mediante retinopexia combinada con vitrectomía pars plana. Resultados: La recuperación visual mejor o igual que logMAR 0,397 (20/50) se dió en 41,7% de ojos y 16,7%. alcanzaron agudeza visual logMAR 0,301 (20/40). Cinco ojos no alcanzaron una ganancia de líneas de visión mayor a cinco. El líquido submacular ausente se observó en la mayoría de ojos que recuperaron más de cinco líneas al igual que aquellos con elipsoide conservado. La regularidad del neuroepitelio y el edema en el periodo posquirúrgico no mostraron comportamientos claros respecto a recuperación visual al igual que la altura del desprendimiento y el número de cuadrantes afectados. Una mejor recuperación visual fue más frecuente en aquellos con menos de cinco semanas de desprendimiento de retina. Conclusiones: El retraso menor a cinco semanas en la resolución del desprendimiento de retina, la conservación del elipsoide y la ausencia de líquido submacular en el periodo postquirúrgico se observó más frecuentemente en ojos con mejor recuperación visual.

Influence of rurality, deprivation and distance from clinic on uptake in men invited for abdominal aortic aneurysm screening

Acknowledgements This study received no specific funding. The study involved the analysis of data collected routinely as part of the national AAA screening programme in Scotland.

Effectiveness and Cost-effectiveness of Outpatient Physiotherapy After Knee Replacement for Osteoarthritis: Study Protocol for a Randomised Controlled Trial

Background: Primary total knee replacement is a common operation that is performed to provide pain relief and restore functional ability. Inpatient physiotherapy is routinely provided after surgery to enhance recovery prior to hospital discharge. However, international variation exists in the provision of outpatient physiotherapy after hospital discharge. While evidence indicates that outpatient physiotherapy can improve short-term function, the longer term benefits are unknown. The aim of this randomised controlled trial is to evaluate the long-term clinical effectiveness and cost-effectiveness of a 6-week group-based outpatient physiotherapy intervention following knee replacement. Methods/design: Two hundred and fifty-six patients waiting for knee replacement because of osteoarthritis will be recruited from two orthopaedic centres. Participants randomised to the usual-care group (n = 128) will be given a booklet about exercise and referred for physiotherapy if deemed appropriate by the clinical care team. The intervention group (n = 128) will receive the same usual care and additionally be invited to attend a group-based outpatient physiotherapy class starting 6 weeks after surgery. The 1-hour class will be run on a weekly basis over 6 weeks and will involve task-orientated and individualised exercises. The primary outcome will be the Lower Extremity Functional Scale at 12 months post-operative. Secondary outcomes include: quality of life, knee pain and function, depression, anxiety and satisfaction. Data collection will be by questionnaire prior to surgery and 3, 6 and 12 months after surgery and will include a resource-use questionnaire to enable a trial-based economic evaluation. Trial participation and satisfaction with the classes will be evaluated through structured telephone interviews. The primary statistical and economic analyses will be conducted on an intention-to-treat basis with and without imputation of missing data. The primary economic result will estimate the incremental cost per quality-adjusted life year gained from this intervention from a National Health Services (NHS) and personal social services perspective. Discussion: This research aims to benefit patients and the NHS by providing evidence on the long-term effectiveness and cost-effectiveness of outpatient physiotherapy after knee replacement. If the intervention is found to be effective and cost-effective, implementation into clinical practice could lead to improvement in patients’ outcomes and improved health care resource efficiency.

Survivorship and Clinical Outcome of the Minimally Invasive Uniglide Medial Fixed Bearing, All-polyethylene Tibia, Unicompartmental Knee Arthroplasty at a Mean Follow-up of 7.3 Years

Background: Medial UKA performed in England and Wales represents 7 to 11% of all knee arthroplasty procedures, and is most commonly performed using mobile-bearing designs. Fixed bearing eliminates the risk of bearing dislocation, however some studies have shown higher revision rates for all-polyethylene tibial components compared to those that utilize metal-backed implants. The aim of the study is to analyse survivorship and maximum 8-year clinical outcome of medial fixed bearing, Uniglide unicompartmental knee arthroplasty performed using an all-polyethylene tibial component with a minimal invasive approach. Methods: Between 2002 and 2009, 270 medial fixed UKAs were performed in our unit. Patients were reviewed pre-operatively, 5 and 8 years post-operatively. Clinical and radiographic reviews were carried out. Patients’ outcome scores (Oxford, WOMAC and American Knee Score) were documented in our database and analysed. Results: Survival and clinical outcome data of 236 knees with a mean 7.3 years follow-up are reported. Every patient with less than 4.93 years follow-up underwent a revision. The patients’ average age at the time of surgery was 69.5 years. The American Knee Society Pain and Function scores, the Oxford Knee Score and the WOMAC score all improved significantly. The 5 years survival rate was 94.1% with implant revision surgery as an end point. The estimated 10 years survival rate is 91.3%. 14 patients were revised before the 5 year follow-up. Conclusion: Fixed bearing Uniglide UKA with an all-polyethylene tibial component is a valuable tool in the management of a medial compartment osteoarthritis, affording good short term survivorship.

Improving Patients’ Experience and Outcome of Total Joint Replacement: the RESTORE Programme

BACKGROUND: Total hip replacements (THRs) and total knee replacements (TKRs) are common elective procedures. In the REsearch STudies into the ORthopaedic Experience (RESTORE) programme, we explored the care and experiences of patients with osteoarthritis after being listed for THR and TKR up to the time when an optimal outcome should be expected. OBJECTIVE: To undertake a programme of research studies to work towards improving patient outcomes after THR and TKR. METHODS: We used methodologies appropriate to research questions: systematic reviews, qualitative studies, randomised controlled trials (RCTs), feasibility studies, cohort studies and a survey. Research was supported by patient and public involvement. RESULTS: Systematic review of longitudinal studies showed that moderate to severe long-term pain affects about 7–23% of patients after THR and 10–34% after TKR. In our cohort study, 10% of patients with hip replacement and 30% with knee replacement showed no clinically or statistically significant functional improvement. In our review of pain assessment few research studies used measures to capture the incidence, character and impact of long-term pain. Qualitative studies highlighted the importance of support by health and social professionals for patients at different stages of the joint replacement pathway. Our review of longitudinal studies suggested that patients with poorer psychological health, physical function or pain before surgery had poorer long-term outcomes and may benefit from pre-surgical interventions. However, uptake of a pre-operative pain management intervention was low. Although evidence relating to patient outcomes was limited, comorbidities are common and may lead to an increased risk of adverse events, suggesting the possible value of optimising pre-operative management. The evidence base on clinical effectiveness of pre-surgical interventions, occupational therapy and physiotherapy-based rehabilitation relied on small RCTs but suggested short-term benefit. Our feasibility studies showed that definitive trials of occupational therapy before surgery and post-discharge group-based physiotherapy exercise are feasible and acceptable to patients. Randomised trial results and systematic review suggest that patients with THR should receive local anaesthetic infiltration for the management of long-term pain, but in patients receiving TKR it may not provide additional benefit to femoral nerve block. From a NHS and Personal Social Services perspective, local anaesthetic infiltration was a cost-effective treatment in primary THR. In qualitative interviews, patients and health-care professionals recognised the importance of participating in the RCTs. To support future interventions and their evaluation, we conducted a study comparing outcome measures and analysed the RCTs as cohort studies. Analyses highlighted the importance of different methods in treating and assessing hip and knee osteoarthritis. There was an inverse association between radiographic severity of osteoarthritis and pain and function in patients waiting for TKR but no association in THR. Different pain characteristics predicted long-term pain in THR and TKR. Outcomes after joint replacement should be assessed with a patient-reported outcome and a functional test. CONCLUSIONS: The RESTORE programme provides important information to guide the development of interventions to improve long-term outcomes for patients with osteoarthritis receiving THR and TKR. Issues relating to their evaluation and the assessment of patient outcomes are highlighted. Potential interventions at key times in the patient pathway were identified and deserve further study, ultimately in the context of a complex intervention.

Soft tissue sarcoma: biology and therapeutic correlates

Soft tissue sarcomas (STS) comprise a heterogenenous group of greater than 50 malignancies of putative mesenchymal cell origin and as such they may arise in diverse tissue types in various anatomical locations throughout the whole body. Collectively they account for approximately 1% of all human malignancies yet have a spectrum of aggressive behaviours amongst their subtypes. They thus pose a particular challenge to manage and remain an under investigated group of cancers with no generally applicable new therapies in the past 40 years and an overall 5-year survival rate that remains stagnant at around 50%. From September 2000 to July 2006 I undertook a full time post-doctoral level research fellowship at the MD Anderson Cancer Center, Houston, Texas, USA in the department of Surgical Oncology to investigate the biology of soft tissue sarcoma and test novel anti- sarcoma adenovirus-based therapy in the preclinical nude rat model of isolated limb perfusion against human sarcoma xenografts. This work, in collaboration with colleagues as indicated herein, led to a number of publications in the scientific literature furthering our understanding of the malignant phenotype of sarcoma and reported preclinical studies with wild-type p53, in a replication deficient adenovirus vector, and oncolytic adenoviruses administered by isolated limb perfusion. Additional collaborative and pioneering preclinical studies reported the molecular imaging of sarcoma response to systemically delivered therapeutic phage RGD-4c AAVP. Doxorubicin chemotherapy is the single most active broadly applicable anti-sarcoma chemotherapeutic yet only has an approximate 30% overall response rate with additional breakthrough tumour progression and recurrence after initial chemo-responsiveness further problematic features in STS management. Doxorubicin is a substrate for the multi- drug resistance (mdr) gene product p-glycoprotein drug efflux pump and exerts its main mode of action by induction of DNA double-strand breaks during the S-phase of the cell cycle. Two papers in my thesis characterise different aspects of chemoresistance in sarcoma. The first shows that wild-type p53 suppresses Protein Kinase Calpha (PKCα) phosphorylation (and activation) of p-glycoprotein by transcriptional repression of PKCα through a Sp-1 transcription factor binding site in its -244/-234 promoter region. The second paper demonstrates that Rad51 (a central mediator of homologous recombination repair of double strand breaks) has elevated levels in sarcoma and particularly in the S- G2 phase of the cell cycle. Suppression of Rad51 with small interfering RNA in sarcoma cell culture led to doxorubicin chemosensitisation. Reintroduction of wild-type p53 into STS cell lines resulted in decreased Rad51 protein and mRNA expression via transcriptional repression of the Rad51 promoter through increased AP-2 binding. In light of poor response rates to chemotherapy, escape from local control portends a poor prognosis for patients with sarcoma. Two papers in my thesis characterise aspects of sarcoma angiogenesis, invasion and metastasis. Human sarcoma samples were found to have high levels of matrix metalloproteinase-9 (MMP-9) with expression levels that correlated with p53 mutational status. MMP-9 is known to degrade extracellular collagen, contribute to the control of the angiogenic switch necessary in primary tumour progression and facilitate invasion and metastasis. Reconstitution of wild-type p53 function led to decreased levels of MMP-9 protein and mRNA as well as zymography-assessed MMP-9 proteolytic activity and decreased tumour cell invasiveness. Reintroduction of wild-type p53 into human sarcoma xenografts in-vivo decreased tumour growth and MMP-9 protein expression. Wild-type p53 was found to suppress mmp-9 transcription via decreased binding of NF-κB to its -607/-595 mmp-9 promoter element. Studies on the role of the VEGF165 in sarcoma found that sarcoma cells stably transfected with VEGF165 formed more aggressive xenografted tumours with increased vascularity, growth rate, metastasis, and resistance to chemotherapy. Use of the anti-VEGFR2 antibody DC101 enhanced doxorubicin sensitivity at sub-conventional dosing, inhibited tumour growth, decreased development of metastases, and reduced tumour micro-vessel density while increasing the vessel maturation index. These effects were explained primarily through effects on endothelial cells (e.c.s), rather than the tumour cells per se, where DC101 induced e.c. sensitivity to doxorubicin and suppressed e.c. production of MMPs. The p53 tumour suppressor pathway is the most frequently mutated pathway in sarcoma. Recapitulation of wild-type p53 function in sarcoma exerts a number of anti-cancer outcomes such as growth arrest, resensitisation to chemotherapy, suppression of invasion, and attenuation of angiogenesis. Using a modified nude rat-human sarcoma xenograft model for isolated limb perfusion (ILP) delivery of wild-type p53 in a replication deficient adenovirus vector I showed that functionally competent wild-type p53 could be delivered to and detected in human leiomyosarcoma xenografts confirming preclinical feasibility - although not efficacious due to low transgene expression. Viral fibre modification to express the RGD tripeptide motif led to greater viral uptake by sarcoma cells in vitro (transductional targeting) and changing the transgene promoter to a response element active in cells with active telomerase expression restricted the transgene expression to the tumour intracellular environment (transcriptional targeting). Delivery of the fibre-modified, selectively replication proficient oncolytic adenovirus Ad.hTC.GFP/ E1a.RGD by ILP demonstrated a more robust, and tumour-restricted, transgene expression with evidence of anti-sarcoma effect confirmed microscopically. Collaborative studies using the fibre modified phage RGD-4C AAVP confirmed that systemic delivery specifically, efficiently, and repeatedly targets human sarcoma xenografts, binds to αv integrins in tumours, and demonstrates a durable, though heterogeneous, transgene expression of 1-4 weeks. Incorporation of the Herpes Simplex Virus thymidine kinase (HSVtk) transgene into RGD-4C AAVP permitted CT-PET spatial and temporal molecular imaging in vivo of transgene expression and allowed quantification of tumour metabolic activity both before and after interval administration of a systemic cytotoxic with predictable and measurable response to treatment before becoming apparent clinically. These papers further the medical and scientific community’s understanding of the biology of soft tissue sarcoma and report preclinical studies with novel and promising anti- sarcoma therapeutics.

Metabolomics as a tool to explore the staphylococcal biofilm

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

An investigation into the role of interleukin-6 in linking systemic and local inflammatory responses in patients with colorectal cancer

Colorectal cancer is the second most common cause of cancer death in the UK. It is accepted that both tumour and host factors are important determinants of disease progression and survival. While systemic and local inflammatory responses are increasingly recognized to be of particular importance the understanding of the mechanisms linking these important inflammatory processes remains unclear. This thesis examines the prognostic importance of measures of systemic and local inflammation and proposes a hypothesis for a link between tumour necrosis, systemic and local inflammatory responses in patients with colorectal cancer. Chapter 3 reports the comparison of the prognostic value of longitudinal measurements of systemic inflammation in patients undergoing curative resection of colorectal cancer. In Chapter 3 the results demonstrate that there was no significant overall change in either mGPS or NLR from pre- to post- operatively. This study highlighted the associations between pre- and post- operative mGPS and NLR and T-stage (p<0.001), TNM stage (p<0.005) and cancer-specific survival. The relationships between pre-operative measurements were examined using multivariate analysis. For pre-operative measurement both mGPS and NLR were associated with cancer-specific survival while when post-operative measures were examined only mGPS was specifically associated with cancer-specific survival (HR 4.81, CI 2.13-10.83, P<0.001). Chapter 4 examines the prognostic value of the Klintrup-Makinen scoring method and the existing limitations with regard to its clinical utility. An automated scoring method using commercially available image analysis software was developed and compared with manual scoring of tumour inflammatory infiltrates. This study demonstrated that both manual K-M scoring (p<0.001) and automated K-M scoring (p<0.05) had prognostic value in patients who had undergone potentially curative resection of colorectal cancer, and that the novel automated method may provide an objective method of assessment of tumour inflammatory infiltrates using routinely stained haematoxylin and eosin sections of tumour samples. In chapter 5 a hypothesis was proposed that Interleukin-6 may link tumour necrosis and systemic and local inflammatory responses in patients with colorectal cancer. This chapter examined the basis for this hypothesis, which is presented in figure 5.1. In addition, in chapter 5 the importance of this potential link is examined. In chapter 6, the hypothesis outlined in chapter 5 was examined in a cohort of patients who had undergone attempted curative resection of colorectal cancer. This study examined the inter-relationships between circulating mediators, in particular IL-6, tumour necrosis and systemic and local inflammatory responses. This results of this study demonstrated that IL-6 was associated with tumour necrosis (<0.001) and mGPS (<0.001) independent of T-stage. Thus adding weight to the hypothesis that elevated circulating concentrations of IL-6 may play a role in modulating both the systemic and local inflammatory responses in patients with cancer. Chapter 7 further develops the hypothesis that IL-6 signalling may be important in modulating systemic and local inflammatory responses in patients with colorectal cancer. Further, in chapter 7 the basis for the role of trans-signalling in this signaling pathway was examined. In this study, we reported that neither expression of the soluble IL-6 receptor or soluble gp130 were associated with systemic or local inflammatory responses. As a result the possible reasons for these findings were explored and future work suggested. A prospective database of patients undergoing attempted curative resection of colorectal cancer in Glasgow Royal Infirmary was used throughout this thesis. This database was created and is maintained regularly by successive research fellows at the Royal Infirmary. The work presented in this thesis highlights the importance of the host response in the form of systemic and local inflammation in patients with colorectal cancer and proposes a link between these responses and tumour necrosis. In addition, this work adds weight to the body of evidence suggesting that assessment of these host responses may improve stratification to treatment for patients with colorectal cancer. Further, this work proposes a mechanistic link, between tumour necrosis, systemic and local inflammatory responses through Interleukin-6, that merits further investigation.

Improving the assessment and outcome of free tissue transfer breast reconstruction

Introduction: Free tissue transfer using an abdominal tissue flap is a commonly used method of breast reconstruction. However, there are well recognised complications including venous congestion, fat necrosis and flap loss associated with the perfusion of these flaps. Post-operative aesthetic outcome assessment of such breast reconstructions have also proven to be difficult with current methods displaying poor inter-rater reliability and patient correlation. The aim of this research was to investigate potential improvements to the post-operative outcome of free abdominal tissue transfer breast reconstruction by assessing the effects of vascular augmentation interventions on flap perfusion and to assess the use of real-time digital video as a post-operative assessment tool. Methods: An in-vivo pilot study carried out on 12 patients undergoing DIEP flap breast reconstruction assessed the effect on Zone IV perfusion, using LDI and ICG angiography, of vascular augmentation of the flap using the contralateral SIEA and SIEV. A further animal experimental study was carried out on 12 Sprague Dawley rats to assess the effects on main pedicle arterial blood flow and on Zone I and Zone IV perfusion of vascular augmentation of the abdominal flap using the contralateral vascular system. A separate post-operative assessment study was undertaken on 35 breast reconstruction patients who evaluated their own reconstructions via patient questionnaire and underwent photograph and real-time digital video capture of their reconstructions with subsequent panel assessment. Results: Our results showed that combined vascular augmentation of DIEP flaps, using both the SIEA and SIEV together, led to an increase in Zone IV perfusion. Vascular augmentation of the rat abdominal flaps also led to a significant increase in Zone I/IV perfusion, but the augmentation procedure resulted in a decreased main pedicle arterial blood flow. Our post-operative assessment study revealed that real-time digital video footage led to greater inter-rater agreement with regards to cosmesis and shape than photography and also correlated more with patient self-assessment. Conclusion: Vascular augmentation of abdominal free tissue flaps using the contralateral vascular system results in an increase to Zone IV perfusion, however this may lead to decreased main pedicle arterial blood flow. Real-time digital video is a valid post-operative aesthetic assessment method of breast reconstruction outcome and is superior to static photography when coupled with panel assessment.

The role of cancer related inflammation, Src family kinases and matrix metalloproteinase 9 in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer in the UK with 41,000 new cases diagnosed in 2011. Despite undergoing potentially curative resection, a significant amount of patients develop recurrence. Biomarkers that aid prognostication or identify patients who are suitable for adjuvant treatments are needed. The TNM staging system does a reasonably good job at offering prognostic information to the treating clinician, but it could be better and identifying methods of improving its accuracy are needed. Tumour progression is based on a complex relationship between tumour behaviour and the hosts’ inflammatory responses. Sustained tumour cell proliferation, evading growth suppressors, resisting apoptosis, replicative immortality, sustained angiogenesis, invasion & metastasis, avoiding immune destruction, deregulated cellular energetics, tumour promoting inflammation and genomic instability & mutation have been identified as hallmarks. These hallmarks are malignant behaviors are what makes the cell cancerous and the more extreme the behaviour the more aggressive the cancer the more likely the risk of a poor outcome. There are two primary genomic instability pathways: Microsatellite Instability (MSI) and Chromosomal Instability (CI) also referred to as Microsatellite Stability (MSS). Tumours arising by these pathways have a predilection for specific anatomical, histological and molecular biological features. It is possible that aberrant molecular expression of genes/proteins that promote malignant behaviors may also act as prognostic and predictive biomarkers, which may offer superior prognostic information to classical prognostic features. Cancer related inflammation has been described as a 7th hallmark of cancer. Despite the systemic inflammatory response (SIR) being associated with more aggressive malignant disease, infiltration by immune cells, particularly CD8+ lymphocytes, at the advancing edge of the tumour have been associated with improved outcome and tumour MSI. It remains unknown if the SIR is associated with tumour MSI and this requires further study. The mechanisms by which colorectal cancer cells locally invade through the bowel remain uncertain, but connective tissue degradation by matrix metalloproteinases (MMPs) such as MMP-9 have been implicated. MMP-9 has been found in the cancer cells, stromal cells and patient circulation. Although tumoural MMP-9 has been associated with poor survival, reports are conflicting and contain relatively small sample sizes. Furthermore, the influence of high serum MMP-9 on survival remains unknown. Src family kinases (SFKs) have been implicated in many adverse cancer cell behaviors. SFKs comprise 9 family members BLK, C-SRC, FGR, FYN, HCK, LCK, LYN, YES, YRK. C-SRC has been the most investigated of all SFKs, but the role of other SFKs in cellular behaviors and their prognostic value remains largely unknown. The development of Src inhibitors, such as Dasatinib, has identified SFKs as a potential therapeutic target for patients at higher risk of poor survival. Unfortunately, clinical trials so far have not been promising but this may reflect inadequate patient selection and SFKs may act as useful prognostic and predictive biomarkers. In chapter 3, the association between cancer related inflammation, tumour MSI, clinicopathological factors and survival was tested in two independent cohorts. A training cohort consisting of n=182 patients and a validation cohort of n=677 patients. MSI tumours were associated with a raised CRP (p=0.003). Hypoalbuminaemia was independently associated with poor overall survival in TNM stage II cancer (HR 3.04 (95% CI 1.44 – 6.43);p=0.004), poor recurrence free survival in TNM stage III cancer (HR 1.86 (95% 1.03 – 3.36);p=0.040) and poor overall survival in CI colorectal cancer (HR 1.49 (95% CI 1.06 – 2.10);p=0.022). Interestingly, MSI tumours were associated with poor overall survival in TNM stage III cancer (HR 2.20 (95% CI 1.10 – 4.37);p=0.025). In chapter 4, the role of MMP-9 in colorectal cancer progression and survival was examined. MMP-9 in the tissue was assessed using IHC and serum expression quantified using ELISA. Serum MMP-9 was associated with cancer cell expression (Spearman’s Correlation Coefficient (SCC) 0.393, p<0.001)) and stromal expression (SCC 0.319, p=0.002). Serum MMP-9 was associated with poor recurrence-free (HR 3.37 (95% CI 1.20 – 9.48);p=0.021) and overall survival (HR 3.16 (95% CI 1.22 – 8.15);p=0.018), but tumour MMP-9 was not survival or MSI status. In chapter 5, the role of SFK expression and activation in colorectal cancer progression and survival was studied. On PCR analysis, although LYN, C-SRC and YES were the most highly expressed, FGR and HCK had higher expression profiles as tumours progressed. Using IHC, raised cytoplasmic FAK (tyr 861) was independently associated with poor recurrence free survival in all cancers (HR 1.48 (95% CI 1.02 – 2.16);p=0.040) and CI cancers (HR 1.50 (95% CI 1.02 – 2.21);p=0.040). However, raised cytoplasmic HCK (HR 2.04 (95% CI 1.11 – 3.76);p=0.022) was independently associated with poor recurrence-free survival in TNM stage II cancers. T84 and HT29 cell lines were used to examine the cellular effects of Dasatinib. Cell viability was assessed using WST-1 assay and apoptosis assessed using an ELISA cell death detection assay. Dasatinib increased T84 tumour cell apoptosis in a dose dependent manner and resulted in reduced expression of nuclear (p=0.008) and cytoplasmic (p=0.016) FAK (tyr 861) expression and increased nuclear FGR expression (p=0.004). The results of this thesis confirm that colorectal cancer is a complex disease that represents several subtypes of cancer based on molecular biological behaviors. This thesis concentrated on features of the disease related to inflammation in terms of genetic and molecular characterisation. MSI cancers are closely associated with systemic inflammation but despite this observation, they retain their relatively improved survival. MMP-9 is a feature of tissue remodeling during inflammation and is also associated with degradation of connective tissue, advanced T-stage and poor outcome when measured in the serum. The lack of stromal quantification due to TMA use rather than full sections makes the value of tumoural MMP-9 immunoreactivity in the prognostication and its association with MSI unknown and requires further study. Finally, SFK activation was also associated with SIR, however, only cytoplasmic HCK was independently associated with poor survival in patients with TNM stage II disease, the group of patients where identifying a novel biomarker is most needed. There is still some way to go before these biomarkers are translated into clinical practice and future work needs to focus on obtaining a reliable and robust scientific technique with validation in an adequately powered independent cohort.