Surgery (RMH) - Theses
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The clinical significance of cyclin E1 deregulation in high grade serous ovarian cancer and basal like breast cancer
High grade serous ovarian cancer (HGSOC) and basal like breast cancer (BLBC) are genomically unstable and aggressive cancers that frequently co-occur and share common molecular features. Of these molecular characteristics are P53 inactivation occurring in almost all cases of HGSOC and BLBC, BRCA1/2 inactivation reported in more than 50% of both cancers and CCNE1 amplification reported in up to 30% and 8% of HGSOC and BLBC respectively. Both HGSOC and BLBC are currently grouped in many clinical trials to test new drugs or drug combination, for instance, PARP inhibitors in the context of BRCA1/BRCA2 mutation. We asked whether deregulated cyclin E1 (CCNE1 amplification and/or its encoding protein, cyclin E1, overexpression) is an additional biomarker that can potentially be used to group patients with both diseases for therapeutic purposes. We studied two well characterised cohorts of 262 HGSOC and 222 familial breast cancer (BLBC enriched) samples of formalin fixed paraffin embedded sections. HGSOC and the BLBC enriched cohort were from patients enrolled in the Australian ovarian (AOCS) and the Kathleen Cuningham Foundation Consortium for research into Familial Breast cancer (KConFab) respectively. Using automated tissue based assay and an in situ hybridization probe that spans 19q12 locus harbouring CCNE1, we assessed the level of CCNE1 amplification. We also assessed the expression of cyclin E1 and a cyclin E1 degradation associated protein FBXW7 and a cyclin E1 deubiquitinase, USP28, by immunohistochemistry, as possible drivers of high cyclin E1 expression in amplified and non-amplified cyclin E1hi subsets. We also assessed the expression of URI1 in our HGSOC cohort. URI1 is a protein encoded by the URI1 gene which co-localise with CCNE1 on 19q12 locus. In HGSOC, we identified seemingly two separate subsets of cyclin E1hi tumors that have different pathological and biological characteristics as well as different clinical outcomes. These are the amplified/cyclin E1hi group that had amplification and high expression of cyclin E1, low expression of FBXW7, higher genomic instability, intact BRCA1/2 and worse outcome. The other is the non-amplified/cyclin E1hi tumors that typically had high expression of cyclin E1 in the absence of amplification, high USP28 expression, lower genomic instability, more prevalent BRCA1/2 loss and more favorable outcome compared to the amplified group. Next we assessed cyclin E1 deregulation in the overlapping groups BRCA1 mutant breast cancer and BLBC. Both subtypes had significantly higher expression of cyclin E1 and amplification compared to other breast cancer types. However, the intensity of cyclin E1 expression and level of 19q12 amplification were lower in BLBC compared to those observed in HGSOC. Moreover, in BRCA1 mutant breast cancer and BLBC patients, only high expression of cyclin E1 was associated with lower overall survival while amplification did not seem to impact outcome. These observations were further supported by our meta-analysis that included our cohorts as well as other published datasets. In the meta-analysis, both CCNE1 amplification and cyclin E1 expression were found to be adverse prognostic factors in HGSOC while only high expression was associated with worse survival in BLBC patients. In fact, both amplified and non-amplified cyclin E1hi BLBC subsets shared almost all cyclin E1 deregulation associated features as well as many features with the non-amplified cyclin E1hi HGSOC subset. Of these are the prevalence of high expression of the cyclin E1 deubiquitinase, USP28, BRCA inactivation, the lower genomic instability and cyclin E1hi linked adverse outcome. In order to provide better therapeutic options for cyclin E1hi BRCA1 mutant breast cancer/BLBC patients, we sought to further assess mechanisms behind the co-occurrence of cyclin E1 overexpression and BRCA1 inactivation. Using the KConFab cohort we have found that BRCA1 loss correlated with decreased phosphorylation of cyclin E1, on Threonine 62, assessed by immunohistochemistry. We also showed by in vitro analysis that BRCA1 loss in cell lines led to cell cycle specific stabilisation of cyclin E1 by reducing cyclin E1 T62 phosphorylation. Conversely, BRCA1 overexpression increased T62 phosphorylation. Overexpression of cyclin E1 with an inactivated T62 site, to mimic loss of phosphorylation, increased cyclin E1 stability and resistance to Paclitaxel. These findings suggest that BRCA1 regulates cyclin E1 stability in breast cancer cells via regulating T62 phosphorylation. We next assessed a combination therapy that target cyclin E1 and BRCA1 inactivation using CDK2 and PARP inhibitors. CDK inhibitors are suggested to induce DNA damage and therefore we hypothesised that CDK2 inhibition would enhance sensitivity of BRCA1 deficient cells to PARP inhibition. Our finding is that CDK2 inhibition induced DNA damage and synergised with the PARP inhibitor Rucaparib in BRCA1 mutated cell lines. Combination treatment of xenograft are in progress but the preliminary data is supportive of our hypothesis. Our results propose a new therapeutic strategy for BRCA1-mutant breast cancer/BLBC by combining CDK2 and PARP inhibitors to enhance synthetic lethality. As this group shares similarities with non-amplified cyclin E1hi HGSOC subset, we suggest that this combination is likely to be effective in the comparable HGSOC subset.
The Postoperative Quality of Recovery Score: validation of its cognitive domain and feasibility analysis of its use in an interventional trial and in providing individualised real-time recovery data
“Knowledge is power. Information is liberating.” Kofi Atta Annan (1998). Modern postoperative recovery assessment has progressed from that which was focused purely on the physiological restitution in the immediate postoperative period to one that is multidimensional, individualised, dichotomised and provided in real time. The Postoperative Quality of Recovery Score (PostopQRS) is an extensively validated multidimensional recovery assessment tool that has been developed de-novo for assessment of recovery in postoperative patients, and has been widely adopted as an outcome measure in observational trials. It is unique in its ability to provide individualised recovery information to each patient, and in its assessment of a patient’s postoperative recovey in relation to their own unique preoperative baseline performance. Through its digital interface, it has the potential to provide individualized, contemporaneous recovery data to each patient, thus keeping patients informed of their own recovery throughout their postoperative journey, and thus potentially improving their ultimate postoperative outcome. Prior to this thesis, the PostopQRS’ cognitive domain, whilst based on widely accepted neurocognitive tests, was yet to demonstrate clinical face validity in its assessment of cognitive recovery, and was currently unable to assess cognitive recovery in patients who score low on preoperative cognitive baseline testing. Simmilarly, the PostopQRS had been widely adopted as an outcome measure in observational trials but was yet to be implemented in interventional studies. Furthermore, the utility of the PostopQRS in providing real-time recovery assessment was yet to be fully explored. The EchoNOF-I pilot study demonstrated the clinical utility of the PostopQRS as an outcome measure in the interventional trial setting, and demonstrated feasibility with providing fractured neck of femur surgery patients with focused point of care ultrasound. This study highlighted the need for there to be a validated method with which to measure cognitive recovery in patients with low cognitive baseline PostopQRS scoring. Head-to-head comparison of patient cognitive performance on both the PostopQRS and formal neurocognitive test battery was performed, both at preoperative baseline and during postoperative recovery. This demonstrated clinical face validity in defining patients as having low, as opposed to normal, PostopQRS baseline performance, and demonstrated face validity in the proposed method with which to score cognitive recovery in thse patients with low cognitive baseline scoring. The RTR-I pilot study was ground-breaking as it was the first study in which the PostopQRS was used as the intervention itself, and which demonstrated the clinical feasibility of the PostopQRS in providing patients with individualised real-time recovery information throughout their recovery journey. This study has the potential to revolutionise recovery assessment from one that has been traditionally research focused, to one that has direct clinical applications that may ultimately improve patient outcomes postoperatively. Work from this thesis has formed the basis for three additional multicentre randomised control trials (RTR-II, ECHONOF –II and ECHONOF –III), one MACH (Translational Research Projects 2019), one ANZCA (ANZCA Projects 2019) and two NHMRC grant applications (Project Grants 2019, MRFF Keeping Australians Out of Hospital 2019). The modified scoring system with which to measure cognitive recovery in patients with low cognitive baseline PostopQRS scoring has been adopted into the PostopQRS.com website. What is truly exciting is the future post-doctoral exploration of the potential for the PostopQRS to provide individualised real-time recovery information to both patient and health care provider, and through this, ultimately improve patient outcomes.
The Genomics of Oral and Oropharyngeal Squamous Cell Carcinoma
Head and neck squamous carcinomas (HNSCCs) are a diverse group of squamous cell cancers in the upper aerodigestive tract. Whilst tobacco, alcohol, and the human papilloma virus (HPV) have been implicated in HNSCC oncogenesis, there is an incomplete understanding of how these factors impact upon molecular characteristics. In this study, we aim to better understand the genomic characteristics of HNSCC and the role of these risk factors in tumour development. Data for 528 patients from the Cancer Genome Atlas (TCGA) was used for in silico analyses to delineate the role of HPV in oral cavity cancers, where evidence for viral-induced oncogenesis remains contentious. A clear mutational signature for viral oncogenesis was identified in oral cavity carcinomas, as well as corresponding signatures in gene expression and DNA methylation, indicative of a role for HPV in the formation of oral cavity cancers. TCGA patients with and without tobacco and alcohol risk factors - non-smokers/non-drinkers (NSND) – were evaluated. Unfortunately, the proportion of NSND patients in the TCGA was insufficient for adequate analysis. Targeted next-generation sequencing for 69 genes as well as 4 HPV subtypes was therefore carried out on 186 patients recruited from our hospital. As frozen tissue was not available for all patients, a workflow to sequence and analyse formalin fixed paraffin embedded (FFPE) samples was developed. Differential mutation of 5 different genes was identified in the NSND group, more than for any other clinicopathologic variable. A high rate of mutations in the extra-cellular domain of NOTCH1 was noted in the mutational data. The role of these putatively inactivating NOTCH1 mutations in HNSCC remains uncertain. NOTCH1 knockouts were induced in HNSCC cell lines using the CRISPR/Cas9 gene editing system to assess the role of these mutations. No phenotypical differences could be identified using in vitro assays. Transcriptomic analysis of the edited cell lines identified alterations in gene expression in cellular adhesion pathways, consistent with epithelial-mesenchymal transition and the known activity of the Notch pathway. Overall, it appears that inactivating mutations in NOTCH1 may result in a paradoxical activation of the Notch pathway. In conclusion, we have established that differing exposures to risk factors for HSNCC result in molecular differences. These differences require validation and in-depth exploration. HPV has also been implicated in the development of oral cavity carcinomas, and novel treatment protocols for HPV-positive oropharyngeal cancers should also be considered for patients with these cancers. The prominence of the Notch pathway in HNSCC is notable, and the apparent paradoxical role of NOTCH1 mutations on pathway function deserves further follow up.
Use of administrative data to create a colorectal cancer database
Background: Research into Colorectal cancer (CRC) require maintenance of clinical cancer databases with complex datasets. These are resource intensive, region specific, and compromised by reporting bias . Administrative data are routinely captured for each hospital admission and may serve as an alternative source for populating databases. However, the accuracy of administrative data has not been fully explored and may vary by data item. The aims of this study included identifying a cohort of new CRC patients from administrative data, measuring its accuracy, and deriving coding algorithms to improve the accuracy of diagnoses, procedures and short-term outcomes. There has been much debate that major surgery, in particular for cancer patients, should be concentrated in tertiary centres, based on the premise that high volume centres achieve better outcomes. In this study, we investigated two hypotheses: that the majority of complex colorectal cancer resections are performed in major city hospitals and that the short-term outcomes are better in CSSANZ (Colorectal Surgical Society of Australia and New Zealand) hospitals. Large Inpatient administrative databases are a common source used to identify comorbidities recorded with International Classification of Disease (ICD) diagnostic codes. These data sources may be used to assess the effect of baseline comorbidity status on surgical care outcomes. In this study, we hypothesized that the ASA PS (American Society of Anaesthesiologists physical status) classification can predict short-term outcomes after a colorectal cancer resection when compared to the Elixhauser comorbidity index (ECI). Methods: A retrospective study was conducted to identify all new colorectal cancer resections at The Royal Melbourne Hospital from 1st of January 2008 to 31st of December 2013, using administrative data. Code combinations and algorithms were used to improve the accuracy of administrative data. These algorithms were utilized to identify an accurate cohort of colorectal cancer resection cases from the Victorian Admitted Episodes Dataset (VAED), between July 2008 to June 2013. The short-term outcomes and workloads were compared in public hospitals across the state of Victoria. The algorithms constructed were also utilised to identify an accurate cohort of CRC resection cases from Dr Foster Global Comparators Victorian dataset. ASA PS classification scores were identified from these cases. Multiple linear regression models were constructed to study the association between comorbidity indices and short-term outcomes. Results: It is possible to use administrative data to identify new colorectal cancer patients who have had a surgical resection, using specific coding algorithms. Administrative data has an accuracy of 80-100% for most data fields, and this accuracy can be improved using coding algorithms. An accurate cohort of colorectal cancer resection cases was identified from the VAED dataset. Seventy-three percent of CRC resections in the state were performed in metropolitan city hospitals. There was no significant difference in LOS (length of stay), mortality and reoperation rates between CSSANZ and non-CSSANZ hospitals. This study demonstrates that administrative data is both cost-effective and informative. The ASA PS model was indeed shown to be a strong predictor of the primary outcome: length of stay (LOS). The significant predictors of LOS were emergency operations, rectal cancer resections, ASA3 and patients age. The Elixhauser model was a better predictor than the ASA PS model. However, the full model adjusted for both the ECI and ASA PS grade was the best predictor of outcome. The study indeed showed the ability of the ASA PS classification to identify short-term clinical outcomes. Conclusion: These studies make the possibility of a Victorian CRC registry containing all surgical CRC patients a real possibility. Such a registry would enable outcomes research across the whole state with the possibility of data linkage to international administrative data sets.
Hormonally induced defects of DNA damage repair genes: an oncogenic driver of prostate cancer
Patients with defects in the mismatch repair pathway, driven by either MSH2 or MSH6 loss, experience a significant increase in the incidence of prostate cancer, while germline mismatch repair defects in either MLH1 or PMS2, exhibit no such increase. This PhD project demonstrates that androgen-receptor activation, a known driver of prostate cancer, can disrupt the MSH2 gene in prostate cancer model systems through the induction of structural variations. Prostate tumours from two contrasting risk cohorts were screened to confirm loss of MSH2 protein expression in a small number of patients. Surprisingly, it was also found that a small but significant fraction of high-risk cases exhibited reduced expression of MSH2 without complete loss. Stratifying a large independent TCGA prostate cancer cohort for MSH2 expression levels revealed that patients whose tumours exhibited either complete loss or aberrant levels of MSH2 had significantly worse survival outcomes and accelerated clinical progression. In contrast, aberrant MSH2 levels had no impact on clinical survival in colorectal cancer. This PhD project also demonstrates that reduced expression of MSH2 can be explained by androgen-induced microRNA regulatory mechanisms. Here, it is demonstrated that miR-21 and miR-141 may both target the MSH2 gene leading to reduced MSH2 protein staining and both microRNAs were seen to be upregulated in prostate cancer patients with reduced MSH2 levels. Interestingly, this thesis also shows that miR-21 and miR-141 are both regulated by androgens, implicating this mechanism as a second androgen driven method of MSH2 downregulation. Importantly this PhD project also found that aberrant MSH2 expression in prostate tumours does not induce the same enhanced immune cell mobilisation seen in colorectal tumours suggesting that the prostate is an immune privileged site. This is contrary to the findings of other studies and may warrant a re-evaluation of whether MSH2 deficient prostate cancers are likely to benefit from immunotherapies. To further investigate if the prostate tumour microenvironment is indeed in an immunosuppressive state, a detailed investigation of the transcriptomic profile of the cells of the tumour microenvironment was also conducted. This resulted in not only the discovery of immunosuppressive signatures in tumour infiltrating T-cells but also significant transcriptomic alterations in other cancer associated pathways such as osteogenesis, cell migration, epithelial mesenchymal transition (EMT), hormone signalling and angiogenesis throughout the tumour microenvironment compared to the cellular make-up of benign prostatic tissue. The data presented in this thesis constitute a significant contribution to the current understanding of how defects in the mismatch repair gene MSH2 may affect prostate cancer severity. Additionally, these studies demonstrate multiple mechanisms through which the prostate tumour microenvironment may enhance prostate cancer progression.
Prostate cancer cell adaptation to profound androgen suppression
Androgen deprivation therapy is the mainstay of treatment for advanced prostate cancer. Although castration results in tumour regression and symptom relief in the majority of cases, the effects are short lived, with most patients demonstrating castration resistant progression within 1-3 years. Numerous molecular mechanisms have been implicated in the development of this resistance to treatment, including maintenance of intraprostatic testosterone levels and changes in the expression of and/or alternative splicing of the androgen receptor. Many of these observations however have been made in cell lines and/or xenograft models, which do not necessarily recapitulate the complex microenvironment in which clinical resistance develops. Whole Genome Sequencing (WGS) and RNASeq has been performed on samples from a cohort of patients who received neo-adjuvant androgen deprivation therapy prior to prostatectomy, and had varying responses, to elucidate the genomic drivers of castration resistant disease. WGS analysis has shown no commonality in neither Single Nucleotide Variations or Copy Number Variations in each response group, nor a distinct change between the pre and post-treatment samples indicating that there is not a selection for a resistant sub clone as a result of androgen deprivation treatment. Analysis of RNA-Seq data has shown no overexpression of the androgen receptor, nor the presence of ARV7. It has however shown a striking difference between treated and untreated samples, displaying a phenotypic change which has occurred as a response to treatment; including an epithelial to mesenchymal transition signature and basal cell signature; as well as an up-regulation in expression of FGF7 and FGF2. Cell line validation assays have indicated that these may be playing a role in treatment persistence in a castrate environment.
Clinical and functional characterisation of novel gene candidates for colorectal cancer
Colorectal cancer (CRC) is the third most common cancer worldwide, affecting over 15,000 individuals in Australia each year. While CRC is often detected at a stage where resection of the primary tumour is possible, approximately 50% will relapse and die from metastatic disease. Current practice to determine clinical management and prognosis is determined by tumour depth (T) and lymph node stage (N) and the extent of cancer spread at diagnosis (M) (TNM staging). However, clinical outcomes of patients with the same TNM staging can be heterogeneous. While adjuvant 5-flurorouracil (5-FU) based chemotherapy is offered to the majority of patients with stage III CRC it is only offered for high risk stage II CRC and many will relapse to 5-FU as a first line therapy. Therefore, there is a need to identify markers to better predict prognosis and better stratify patients with stage II/III CRC for treatment regimes. Currently, the use of biomarkers in prognostication for the management of CRC is still not common practice with only a few markers used in the clinic. The mutational landscape of CRC has revealed large numbers of mutated genes; however, it is not known which are drivers that contribute to carcinogenesis and whether such mutations provide prognostic information. This thesis aims to (I) identify clinically relevant markers and patient subgroups of CRC to better predict prognosis and better stratify patients with stage II/III CRC for treatment regimes, (II) to investigate the potential for a MACROD2 deletion as a novel driver of CRC tumourigenesis and (III) to elucidate the impact of a MACROD2 gene deletion on DNA repair and chromosomal instability in CRC.
Investigating the evolution of structural variation in cancer
Cancers arise from single progenitor cells that acquire mutations, eventually dividing into mixed populations with distinct genotypes. These populations can be estimated by identifying common mutational profiles, using computational techniques applied to sequencing data from tumour tissue samples. Existing methods have largely focused on single nucleotide variants (SNVs), despite growing evidence of the importance of structural variation (SV) as drivers in certain subtypes of cancer. While some approaches use copy-number aberrant SVs, no method has incorporated balanced rearrangements. To address this, I developed a Bayesian inference approach for estimating SV cancer cell fraction called SVclone. I validated SVclone using in silico mixtures of real samples in known proportions and found that clonal deconvolution using SV breakpoints can yield comparable results to SNV-based clustering. I then applied the method to 2,778 whole-genomes across 39 distinct tumour types, uncovering a subclonal copy-number neutral rearrangement phenotype with decreased overall survival. This clinically relevant finding could not have been found using existing methods. To further expand the methodology, and demonstrate its application to low data quality contexts, I developed a novel statistical approach to test for clonal differences in high-variance, formalin-fixed, paraffin-embedded (FFPE) samples. Together with variant curation strategies to minimise FFPE artefact, I applied the approach to longitudinal samples from a cohort of neo-adjuvant treated prostate cancer patients to investigate whether clonal differences can be inferred in highly noisy data. This thesis demonstrates that characterising the evolution of structural variation, particularly balanced rearrangements, results in clinically relevant insights. Identifying the patterns and dynamics of structural variation in the context of tumour evolution will ultimately help improve understanding of common pathways of tumour progression. Through this knowledge, cancers driven by SVs will have clearer prognoses and clinical treatment decisions will ultimately be improved, leading to better patient outcomes.
The role of quiescence in treatment resistance and malignancy in glioblastoma multiforme
Glioblastoma multiforme (GBM) represents the most malignant incarnation of glial tumours – a World Health Organisation (WHO) grade IV brain malignancy. GBM is the most common primary brain tumour in adults, accounting for 78% of all malignant central nervous system (CNS) tumours, and affecting 2-3 people per 100,000 in Europe and North America, with an average survival of only 14.6 months. Despite continued research and incremental advances in imaging, surgery, and chemoradiotherapy, patient survival has stagnated in the past decade, with several promising lines of investigation failing to fully deliver on their anticipated translational outcomes. Recent advances in genetic sequencing and computational biology have allowed the simultaneous comparison of large numbers of patient cancer cell genomes and identified several GBM subtypes. It is hoped that such stratification will one day allow clinicians to tailor treatments specific to each GBM subtype as has already happened in cancers like medulloblastoma. However, despite best efforts, GBM remains highly aggressive, infiltrative, and treatment-resistant, rendering it incurable by current treatment modalities. Invasion of tumour cells into normal brain prohibits a surgical cure, while a high cancer stem cell (CSC) component resists treatment with radiation and temozolomide (TMZ) – both of which are more effective against rapidly dividing cells – and relapse remains the rule. Molecular mechanisms underlie GBM’s treatment resistance, and elucidating the key drivers that garner inherent resistance to the quiescent, stem-like fraction of cells that lead to treatment failure therefore presents as an exciting area of research that may uncover new potential drug targets that improve patient outcomes. This study has shown that the proliferation rate of GBM cells is spectral, approximating a positively skewed normal distribution, with highly proliferative cells at one end and quiescent cells at the other. The quiescent cell fraction was subsequently shown to be inherently more resistant to chemoradiotherapy than the proliferative fraction. The quiescent fraction also displayed increased size, complexity, rates of migration and invasion, secretion of extracellular matrix-degrading enzymes, and invadopodia activity than their proliferative counterparts. Similarly, quiescent cells proliferated slower as intracranial tumours but displayed significantly greater invasive properties than a subset of proliferative cells grown in vivo. mRNA expression analysis revealed the genetic signature that underpins the disparity in proliferation rate between quiescent and proliferative cells, and the putative genes that are responsible for the malignant properties identified in both populations. This body of work has uncovered the inherently dichotomous treatment response of quiescent and rapidly dividing GBM cells, as well as the difference in their abilities to migrate and invade. This study has also shed light on the fundamental molecular mechanisms that are at the root of treatment resistance and malignancy in this disease. It is hoped that this expression signature will help to inform future studies and treatments that target these differences and make GBM less of a death sentence and more of manageable, chronic disease.
The impact of intensivist-performed, examination extended ultrasound in intensive care medicine
Intensivist-performed ultrasound, including transthoracic echocardiography (TTE) and to a lesser extent lung ultrasound, has been integrated into clinical practice in ICU. However, it is only used when indicated as a goal-directed test to answer a specific clinical question posed by the treating intensivist. The impact of such practice has been shown to be clinically useful and may be associated with better outcome. In contrast, routine practice of ultrasound in critical care is plausible and has a good rationale with diagnostic, monitoring and sometimes therapeutic advantages. In addition to its non-invasive nature, safety and reproducibility, routine use of ultrasound as an extended bedside examination – irrespective of indications – may be clinically useful in various phases during the course of ICU admission. Nonetheless, there are gaps in the literature surrounding this approach. In our literature review, we aim to investigate the role of intensivist-performed critical care ultrasound (TTE and lung with or without diaphragm) on clinical outcomes when performed routinely as an adjunct to clinical assessment during the first 24 hours of admission and during the peri-extubation period as a predicting factor of extubation outcome. The thesis includes three prospective observational studies, where different applications of ultrasound are investigated. Each study is preceded by literature review relevant to the study topic. The first study investigated the feasibility and impact of routine combined limited transthoracic echocardiography and lung ultrasound on diagnosis and management of patients admitted to ICU. Objectives: routine combined limited TTE and lung ultrasound performed within 24 hours of admission to ICU is feasible and changes diagnosis and management in a high proportion of patients. Design: Prospective observational study. Setting: Tertiary ICU. Participants: 93 critically ill participants within 24 hours of admission to ICU. Methods: A treating intensivist documented a clinical diagnosis and management plan before and after combined limited TTE and lung ultrasound. Ultrasound was performed by an independent intensivist and checked for accuracy off-line by a second reviewer. Results: Ultrasound images were interpretable in 99% of cases, with good interobserver agreement. The haemodynamic diagnosis was altered in 66% of participants, comprising new (14%) and altered (25%) abnormal states and exclusion of clinically diagnosed abnormal state (27%). Valve pathology of at least moderate severity was diagnosed for mitral regurgitation (7%), aortic stenosis (1%), aortic stenosis and mitral regurgitation (1%) and tricuspid regurgitation (3%). One case of mitral regurgitation was excluded. Lung pathology diagnosis was changed in 58% of participants, comprising consolidation (13%), interstitial syndrome (4%) and pleural effusion (23%) and exclusion of clinically diagnosed consolidation (6%), interstitial syndrome (3%) and pleural effusion (9%). Management changed in 65% of participants, comprising increased (12%) or decreased (23%) fluid therapy; initiation (10%), change (6%) or cessation (9%) of inotropic, vasoactive or diuretic drugs; non-invasive ventilation (3%); and pleural drainage (2%). Conclusion: Routine screening of patients with combined limited TTE and lung ultrasound on admission to ICU is feasible and frequently alters diagnosis and management. The second study is titled ‘Diaphragmatic regional displacement assessed by ultrasound and correlated to subphrenic organ movement in the critically ill patients - an observational study’. Objectives: The objectives of the study were to identify the most reliably imaged regions of the diaphragm, to evaluate the correlation of movement between different parts of each hemidiaphragm and to assess the agreement between liver or spleen displacement and movement of the ipsilateral hemidiaphragm. Design: Prospective observational study. Setting: Tertiary ICU. Participants: 90 participants Methods: Images of the diaphragm, liver and spleen were obtained using 2-dimensional ultrasound. Acceptable agreement between regions of the diaphragm, liver and spleen was defined as an absence of fixed or proportional bias using Deming regression analysis. Limits of agreement of two standard deviations of the difference less than 30% of the mean value was considered acceptable. Results: We included 90 critically ill participants. The medial (87%) and middle (73%) regions of the right hemidiaphragm, liver (87.7%), medial (71%) and middle (51%) regions of the left hemidiaphragm and spleen (81%) were most frequently imaged. In non-intubated group, acceptable agreement was present between the movements of the middle and medial regions of the right hemidiaphragm, middle and medial regions of the left hemidiaphragm and between the movement of the middle region of the left hemidiaphragm and displacement of the spleen. In the intubated group and in all participants when combined, acceptable agreement was only present between the movements of the middle and medial regions of the right hemidiaphragm and middle and medial regions of the left hemidiaphragm. Acceptable agreement was not present between the diaphragm movement and the liver or spleen displacement in the intubated group or in all participants. Conclusion: The diaphragm medial part is visualised in the majority of studied participants. The medial and middle regions of the hemidiaphragm may be used interchangeably to assess the hemidiaphragm movement. Acceptable agreement does not exist for the diaphragm and solid organ movement, other than for the left middle region and the spleen in non-intubated participants only. The third study is titled ‘The impact of heart, lung and diaphragmatic ultrasound on prediction of failed extubation from mechanical ventilation in critically ill patients: a prospective observational pilot study’. Objectives: Failed extubation from mechanical ventilation in critically ill patients is multifactorial, complex and not well understood. We aimed to identify whether combined TTE, lung and diaphragmatic ultrasound can predict extubation failure in critically ill patients. Design: Prospective observational study. Setting: Tertiary ICU. Participants: 53 participants who were intubated >48 hours and deemed by the treating intensivist ready for extubation. Methods: A 60-minute pre-extubation weaning trial (pressure support <10 cmH2O and positive end expiratory pressure 5 cmH2O) was performed. Prior to extubation, data collected included ultrasound assessment of left ventricular ejection fraction, left atrial area, early diastolic trans-mitral flow velocity wave (E), E/A, E/E', interatrial septal motion, lung loss of aeration score and diaphragm movement. At the end of the weaning trial the rapid shallow breathing index and serum B-type natriuretic peptide concentration were measured. Success and failure of weaning was assessed by defined criteria. Decision to extubate was at the discretion of the treating intensivist. Failure of extubation was defined as re-intubation or non-invasive ventilation within 48 hours after extubation. Results: Of 53 extubated participants, 11 failed extubation. Failed extubation was associated with diabetes, ischaemic heart disease, higher E/E' (OR 1.27, 95% CI 1.05-1.54), left atrial area (OR 1.14, CI 1.02-1.28), fixed rightward curvature of the interatrial septum (OR 12.95, CI 2.73-61.41) and higher loss of aeration score of anterior and lateral regions of the lungs (OR 1.41, CI 1.01-1.82). Conclusion: Failed extubation in mechanically ventilated patients is more prevalent if markers of left ventricular diastolic dysfunction and loss of lung aeration are present. There are encouraging signs that support the use of multiple ultrasound modalities routinely as an adjunct to clinical evaluation of critically ill patients with a strong signal for the use of combined TTE and lung ultrasound. Adding lung ultrasound to TTE is complementary, and enhances the information obtained from TTE. It may also predict outcomes of weaning from mechanical ventilation.
TGF-β signaling regulation in breast cancer metastasis
The majority of cancer fatality is caused by tumor metastasis which is underlined by Epithelial-Mesenchymal-Transition (EMT) and its opposing process Mesenchymal-Epithelial-Transition (MET) at the cellular level. Transforming Growth Factor-β (TGF-β) is one of the main molecular driving forces for EMT. Therefore, more than 20 phase I/II/III clinical trials targeting TGF-β signaling have been developed with mixed outcomes, highlighting the complex nature of TGF-β signaling in driving EMT and metastasis. Bioactive vesicles, exosomes, have recently been an exciting focus of research. An increasing number of publications have shown that exosomes play an essential role in both primary tumor growth and metastatic evolution. However the mechanisms of how exosomes promote cancer malignancy are poorly understood. In this thesis, exosomes were isolated from highly-invasive breast cancer cells MDA-MB-231 and fibroblast NIH3T3 cells by differential centrifugation. We demonstrated that exosomes mediated amplification of TGF-β signaling in highly-invasive breast cancer cells, as well as phenocopying of invasive/metastatic behavior. Targeting exosomes trafficking, secretion and uptake significantly reduced TGF-β responses and consequent cellular functions, indicating a new therapeutic strategy for cancer treatment. Another Transforming Growth Factor-β superfamily member bone morphogenetic proteins (BMPs) inhibit TGF-β-induced EMT and promote MET. However, the mechanism regarding the antagonism and interaction between these two signaling pathways in breast cancer remains unclear. TGF-β abolished BMP-induced Smad1/5 activation in the highly-invasive MDA-MB-231 cells, but to a less extent in the non-invasive MCF7 cells, and even lesser in the normal MCF10A cells. This suggests TGF-β signaling acts in a double whammy fashion in driving cancer invasion and metastasis. We discovered that restored BMP signaling, by combining suboptimal concentration of UO126 and FK506, significantly reduced breast cancer cell self-seeding, liver and bone metastasis in vivo. Surprisingly, no effect on lung metastasis was observed, suggesting a differential role played by BMP signaling in organ specific metastasis. Consequently, there is a potential for them or their analogs to be developed for clinical use. Opposite to EMT, MET is the process whereby disseminated cancer cells re-establish epithelial-like cells and reinitiate proliferative programs. Thus, MET maybe represents a rate-limiting step of metastasis. While a plethora of studies in EMT have been published, more research is needed regarding the MET process and its regulation. In this thesis, a novel modified transwell assay was designed for detecting cell migration as well as detaching-reseeding. Meanwhile, TGF-β signaling activity per cell was measured by infecting MDA-MB-231 cells with Ad-CMV-Gaussia-luc and TGF-β-Smad3 reporter Ad-CAGA-luc. We illustrated that TGF-β plays a positive role in inducing cell migration but a negative one in detaching-reseeding, which may shed new lights for future cancer treatment. Taken together, this study demonstrates that multiple mechanisms may account for the TGF-β-mediated breast cancer cell migration/metastasis, providing substantial evidences for the development of new cancer therapeutics.
Investigation of the role of intra- and peri-prostatic nerves in urinary continence and prostate cancer progression
Background Prostate cancer is a prevalent disease that causes significant morbidity and mortality. Although radical prostatectomy offers excellent disease control, it is associated with significant side effects in some men. One of the most debilitating of these is urinary incontinence. This is usually thought to be due to external urethral sphincter (rhabdosphincter) deficiency. However, the underlying cause of sphincter weakness is not clear. In order to minimise the risk of post-prostatectomy incontinence, clarity of the underlying pathophysiology is required. The neuroanatomy of male urinary continence is incompletely understood. The periprostatic nerves may be involved in the maintenance of normal continence and thus involved in the pathophysiology of post-prostatectomy incontinence. Beyond their fundamental role in tissue innervation, nerves have a complex relationship with pathology. Perineural invasion is an important mechanism for extra-prostatic cancer spread. However, conflicting evidence exists regarding the ability of perineural invasion in a radical prostatectomy specimen to predict disease recurrence. Recently, there is a growing body of evidence to suggest the autonomic nervous system exerts a regulatory effect in cancer (including prostate cancer). Methods A systematic review and meta-analysis was undertaken to investigate if sparing the neurovascular bundle at radical prostatectomy is associated with post-operative urinary continence outcomes. Following from this, three complementary anatomical studies were undertaken to investigate the innervation of the male urethral rhabdosphincter. These were done to investigate potential mechanisms for neurologically mediated post-prostatectomy incontinence. Study methodology included fresh cadaveric dissection, histological evaluation of periprostatic nerve distribution in radical prostatectomy specimens and intraoperative nerve conduction studies. Analysis of prospectively collected data from men treated for clinically localized prostate cancer was interrogated to investigate the relationship between perineural invasion in a radical prostatectomy specimen and clinicopathological variables including biochemical recurrence. More detailed, histological analysis of intraprostatic nerves in radical prostatectomy specimens was undertaken to describe the autonomic nerve subtypes involved in perineural invasion, and those nerves in surrounding normal prostate tissue. These subtype nerve counts were compared against routine clinicopathological variables including recurrence, to evaluate their clinical relevance and determine their role in predicting biochemical recurrence after radical prostatectomy. Results Meta-analysis revealed that sparing the neurovascular bundle during radical prostatectomy is associated with improved continence recovery up to six months post-operatively, but not with long-term outcomes. Fresh cadaveric dissection demonstrated pudendal supply to the male urethral rhabdosphincter, but was neither able to confirm nor exclude the possibility of intrapelvic innervation. Histological evaluation of radical prostatectomy specimens demonstrated the presence of somatic nerves travelling alongside the prostate, which may represent motor fibres destined for the rhabdosphincter. This was supported by evidence from intraoperative neurophysiological studies that demonstrated functional innervation to the male urethral rhabdosphincter travels with the neurovascular bundle in some men. Presence of undifferentiated perineural invasion in radical prostatectomy specimens was not associated with biochemical failure, in the large prospective cohort studied. On histological analysis of intraprostatic nerves in prostate cancer, the majority of nerves were of a mixed bundle type (sympathetic and parasympathetic). Sympathetic non-perineural invasion nerves and non-adrenergic, non-nitrergic perineural invasion nerves and were both found to be independent predictors of biochemical recurrence. Conclusion Nerve sparing should be considered in men with pre-operative erectile dysfunction in the context of appropriate oncological risk stratification, as it may result in improved early continence rates. This thesis supports the hypothesis that in some men, in addition to chief somatic supply via the perineal branch of the pudendal nerve, there exists intrapelvic somatic nerves, which travel in close proximity to the prostate to supply the male urethral rhadbosphincter. Preservation of these intrapelvic somatic continence nerves may provide one explanation for the association between nerve sparing and improved early urinary continence rates. Future research should seek to ascertain the frequency of this anatomical variation in a large cohort in order to inform surgical technique for the optimisation of continence outcomes. The presence of routinely reported perineural invasion at radical prostatectomy does not aid post-operative risk stratification. However, increased density of specific nerve subtypes can be used in predicting recurrence after prostatectomy. This provides further evidence for a relationship between the nervous system and prostate cancer. A greater understanding of the interactions between prostate cancer and nerves will help clarify their role risk stratification and may lead to future therapeutic targets.