Surgery - Doctoral Theses
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Item Exploring the effect of peri-operative inflammation on systemic and local breast cancer biomarkers(University College Cork, 2024) Cullinane, Carolyn; Redmond, Henry Paul; Corrigan, Mark; Connolly, Roisin; Foley, Cathriona; University College CorkBackground Despite curative surgery and optimal treatment, distant metastatic disease can occur in up to 25% of patients with breast cancer. Pre-clinical research has suggested that the surgical insult can promote the growth of new metastases and facilitate seeding of micrometastases into the circulation. The exact mechanism underlying the pro-metastatic potential of the peri-operative period is yet to be fully determined. This thesis aimed to explore the effect of peri-operative inflammation on local and systemic biomarker expression. Hypothesis The peri-operative period can alter local and systemic mediators of breast cancer inflammation and influence circulating tumour DNA (ctDNA)release which may predict clinical and oncological outcomes. Aims 1. To understand the relationship between peri-operative systemic inflammation and breast tumour characteristics, surgical intervention and 30-day morbidity in non-metastatic breast cancer 2. To define the perioperative dynamics of cell free DNA (cfDNA) in breast cancer and explore its prognostic potential. 3. To determine the perioperative dynamics of circulating tumour DNA (ctDNA) in breast cancer and define its prognostic potential. Methods This study is a sub-study of the ICORG 09-07 Breast Cancer Proteomics and Molecular Heterogeneity study, and sample collection is within the remit of the biospecimen protocol (NCT01840293). Patients presenting for elective breast cancer surgery, with non-metastatic disease, aged 18-85 years of age were screened for inclusion prospectively. Blood samples were phlebotomised peri- operatively from patients at the following time-points– pre-operatively the morning of surgery (pre-op), 3-5 hours post-operatively (early post-op), 10-14 days post-operatively (late post-op). Tumour and normal adjacent tumour was collected within 20minutes of surgical resection and cut up into at least 3 pieces (minimum size 2-3mm2 ), and snap frozen in liquid nitrogen separate cryovials. Statistical analysis was performed using IBM SPSS software, version 28 and GraphPad PRISM, version 8.4.2. Graphs were generated using GraphPad PRISM, version 8.4.2. For demographic and clinicopathological data, categorical data were described by their counts and percentages in each. Descriptive analysis was used to depict baseline characteristics. Mann–Whitney U test and Fisher’s test were used to calculate p value in group-comparison analysis. An analysis of variance (ANOVA) was used to compare mean cytokine values peri-operatively followed by multiple comparison analysis using Bonferroni. Student T test or Mann Whitney tests were used to determine differences in peri-operative ctDNA and cfDNA detection. Chi-squared or Fisher’s exact test (ranksum) were carried out to assess associations between ctDNA/cfDNA detection and clinical variables. Binary logistic regression analysis was performed to predict the likelihood of ctDNA positivity based on dichotomous clinical outcomes. Correlation between continuous variables was analysed using Pearson correlation. Correlations were graphically represented using Manhattan plots. The association between cell-free DNA (cfDNA) and DFS were analysed using Mantel Cox log-rank test and Kaplan–Meier curves generated. For all of the above analyses, statistical significance was observed at p <0.5. Results Blood samples were collected from 64 patient’s peri-operatively. There were 38 patients who had their systemic inflammatory mediators analysed peri-operatively. IL-6, IL-8 and IL-10 levels peaked early post-operatively and returned to below baseline levels at 2 weeks post-operatively (p < 0.05). Early post-op IL-6 levels were lower in patients who had breast conserving surgery compared to patients who had a mastectomy (p = <0.01). A significant positive correlation was identified between age and peri-operative IL-6 levels at all time points. Early post-op IL-6 correlated with increasing tumour stage (p = 0.048). When IL6 expression and other IL6 related genes were analysed in normal adjacent to tumour (NAT) and tumour tissue samples, IL6, Leptin, JAK1, CD38, FABP4 and Adiponectin gene expression were significantly higher in NAT compared to matched tumour samples. Pre-operatively the mean cfDNA concentration was 3.82ng/μL (SEM = 0.74). This increased significantly early post-op in keeping with surgical insult to 17.59 ng/μL (SEM = 5.75). A significant linear relationship was observed between early post-op cfDNA concentration and early post-op IL-8 levels (r = 0.430, p = 0.009). Forty-five pre- and post-operative patient samples were suitable for ctDNA analysis. The number of pre-operative ctDNA variants detected correlated positively with pre-operative cfDNA concentration (r = 0.314,p = 0.035). Two patients experienced disease recurrence within the follow up period (median 22 months) and ctDNA was detectable in both their pre- and post-op samples. Kaplan Meier estimates of disease-free survival stratified by the presence of ctDNA did not illustrate any significant difference in DFS. Patients who underwent Axillary Lymph Node Dissection (ALND) had a significant increase in post-op ctDNA variant detection compared to those who underwent SLNB (OR 21.71, p = 0.0063). Conclusion Peri-operative cytokine release appears to correlate with tumour characteristics and the magnitude of surgery. Results from this study demonstrate that cfDNA and ctDNA can be detected peri-operatively using a commercially available ctDNA assay. An increase in post-operative ctDNA burden was evident in patients who underwent more extensive axillary surgery. Longer term follow up is required to determine the prognostic potential of peri-operative inflammatory and ctDNA variant dynamics and if modulation of these parameters might improve patient outcomes.Item The prognostic utility of perioperative blood-based liquid biopsy, inflammatory and tumour biomarkers in patients with non-metastatic breast cancer(University College Cork, 2022) Hassan, Fara; Redmond, Henry Paul; Wang, Jianghuai; Higher Education AuthorityBackground: The analysis of tumour components in blood including systemic mediators of inflammation, tumour marker CA 15-3 and more recently liquid biopsy such as cell free DNA (cfDNA) and circulating tumour DNA (ctDNA) represents an easily accessible, minimally invasive, and cost- effective tool for both the monitoring of breast cancer and assessment of tumour response. These biomarkers in blood of patients with breast cancer have been well documented but largely underexplored in the perioperative period especially in patients with non-metastatic breast cancer. The analysis of circulating nucleic acids in blood, otherwise known as liquid biopsy, has gained significant consideration in the field of breast cancer. Dynamic changes in cfDNA and ctDNA usually correlates with disease status in breast cancer. This thesis investigated the role of blood-based biomarkers including cfDNA and ctDNA, inflammatory biomarkers (IL6, TNF α and VEGF) and tumour marker (CA 15-3) in the preoperative (Preop) and postoperative (Postop) period in patients with non-metastatic breast cancer. Furthermore, the relationship of these biomarkers with tumour characteristics and oncological outcomes in breast cancer was investigated. Methods: A cohort of 62 patients undergoing curative surgery for breast cancer were screened for inclusion. All patients had paired blood samples, preoperatively and post operatively. Post- operative (PO) samples were analysed in either of the following time periods, Postop week 1- 2 (PO week 1-2), Postop week 3-4 (PO week 3-4) and Postop week 5-12 (PO week 5-12). Cell free DNA (cfDNA) was extracted and quantified. Circulating tumour DNA (ctDNA) with PIK3CA gene mutation was detected using HRM PCR and Allele specific fluorescence probe-based PCR. Systemic inflammatory mediators and the tumour marker CA 15-3 were detected using ELISA kit. Results: In the cohort of 62 patients (age, median (IQR), 51.5(45.0-65.0) years), with a median follow- up of 90 months (interquartile range (IQR),60-120 months), significant association was observed between cfDNA concentrations and oncological outcomes in patients with breast cancer. The group of patients who had disease recurrence during the follow up period had significantly higher cfDNA concentrations (cutoff:400 ng/ml) during the perioperative period compared to disease-free patients (Preop and Postop period: p<0.0001). Furthermore, detection of PIK3CA gene mutation in ctDNA in the perioperative period had significant association with oncological outcomes in patients with breast cancer (p<0.0001). In total, 25 (40.3%) and 22 (35%) patients with breast cancer had detectable PIK3CA mutations in ctDNA in Preop and Postop period, respectively. Cox hazard model analysis revealed that PIK3CA mutations in Postop period (hazard ratio (H.R: 18.05, p=0.001) were negative prognostic factor for recurrence free survival (RFS) and overall survival (O.S) (H.R: 11.9, p=0.01) in patients with breast cancer. Postop higher cfDNA concentrations and Postop detection of PIK3CA in ctDNA preceded clinical detection of breast cancer recurrence with an average lead time of 12.00 months (IQR:20-28.5 months) compared to 72.00 (96-120) months; (p<0.0001) for patients who remain disease free on follow up. Univariate and multivariate cox regression analysis indicated that Postop cfDNA concentration and post op detection of PIK3CA (p<0.0001) were negative prognostic factor for RFS and O.S in patients with breast cancer. Serum IL6, TNF α, VEGF and CA15-3 correlated with certain breast tumour characteristics but no =significant effect on survival outcomes except Preop VEGF with significant effect on O.S in breast cancer (p=0.03). Conclusion Research efforts from this thesis have demonstrated potential clinical applications of liquid biopsy with cfDNA concentrations and ctDNA with PIK3CA gene mutation and inflammatory cytokines and CA 15-3 in patients with non-metastatic breast cancer.Item Investigating the utility of blood borne oncological biomarkers in solid tumours: glioma and melanoma(University College Cork, 2021-07-15) Ita, Michael Itak; Lim, Chris; Wang, Jianghuai; Redmond, Henry Paul; University College CorkBloodborne molecular biomarkers are increasingly emerging as significant non-invasive adjuncts to current methods of disease status evaluation in cancer patients. Investigations into the potential utility of these circulating biomarkers as analytic test measures complementing radiological imaging have been occasioned by the invasive nature of malignant tumour tissue biopsy, the need for serial evaluation of tumour burden during therapy, and the need for prognostication. In a series of five studies (four clinical studies and one pre-clinical study), this research work explored the potential utility of plasma cell-free DNA, circulating tumour DNA, cell-free messenger RNA, and bloodborne tumour related proteins as disease biomarkers in patients with glioma and metastatic melanoma. The in vivo research work employed an animal model to study micro RNA mediated epigenetic regulatory mechanisms implicated in therapeutic resistance which is prevalent in melanoma brain metastasis. Specifically, this research work sought to determine whether somatic mutations identified in the plasma samples of patients with glioma were identical or representative of the somatic mutations in synchronously obtained glioma tumour tissue samples. It further sought to determine whether significant differences exist in the plasma transcriptomic profile of glioma patients relative to differences in their tumour characteristics, and also whether any observed differences were representative of synchronously obtained glioma samples and the human cancer genome atlas (TCGA) glioma derived RNA profile. Moreover, this research work explored the relationship between plasma cell-free DNA (cfDNA), serum lactate dehydrogenase (LDH), plasma vascular endothelial growth factor (VEGF), programmed death ligand-1 (PD-L1), interferon-gamma (IFN-γ), and tumour burden in advanced melanoma patients. Furthermore, it sought to examine whether important differences exist in the plasma transcriptomic profile of advanced melanoma patients with a high disease burden compared to patients with a low disease burden or therapeutic response and whether the plasma transcriptomic profile of advanced melanoma patients was representative of TCGA melanoma tumour tissue-derived RNA profile. The methods employed in this research work include; the purification and quantification of circulating cell-free DNA and total RNA from the plasma samples of glioma and metastatic melanoma patients, somatic mutation profiling using DNA derived from FFPE glioma tumour tissue curls and plasma circulating cell-free DNA by amplification refractory mutation system (ARMS®) PCR, pathway-focused gene expression analysis using complementary DNA synthesized from the plasma circulating cell-free messenger ribonucleic acid (ccfmRNA) samples of patients with glioma and advanced melanoma, the extraction and quantification of tumour-related proteins such as LDH, VEGF, PD-L1, and IFN-γ from patients with advanced melanoma by the enzyme-linked immunosorbent assay technique (ELISA), in vivo malignant brain tumour model development, bioluminescence imaging study, immunohistochemistry and microscopy, evaluation of protein expression by flow cytometry, and genomic profiling of total cellular micro RNA using RT- PCR. This research work was able to establish that the detection of plasma circulating tumour DNA originating from the glioma tumour tissues of affected patients is feasible, albeit with a low tumour to plasma mutation concordance. It identified significant differential expression of genes involved in cancer inflammation and immunity crosstalk among patients with different glioma grades, and a positive correlation between the transcriptomic profile of these genes in plasma and tumour samples, and with TCGA glioma derived RNA. Moreover, this research work identified that the incorporation of the quantitative measures of cfDNA, LDH, VEGF and PD-L1 in a suitable multiple regression analysis model was capable of predicting changes in tumour burden in patients with advanced melanoma. Furthermore, it identified and characterized the plasma transcriptomic profile associated with therapeutic response in advanced melanoma patients during immunotherapy. This research work also characterized in a limited way, the tissue and blood markers of therapeutic response and resistance in an in vivo model of melanoma brain metastasis.Item Macrophage polarisation: the impact of M1 versus M2 polarisation on host innate immune responses to bacterial infection(University College Cork, 2015-12) Foley, Niamh M.; Redmond, Henry Paul; Wang, JianghuaiBackground and Aim: Infection is a global burden causing millions of deaths per annum worldwide. In the US sepsis is the tenth leading cause of death with the mortality associated with severe sepsis estimated at 30-50%. Innate immunity is a generic response mediated by the host to protect it from bacterial infection. The recognition of foreign microbes leads to activation of pattern recognition receptors and recruitment of macrophages. In acute bacterial infections, activated macrophages polarised to M1 or M2 states play a major role in the host cytokine response which drives the immune response until the host has overcome the invading microbial pathogen. The aim of this study was to 1) to characterise the cytokine profile of M1 and M2 polarised macrophages 2) to investigate the changes in the cytokine profile of polarised macrophages in response to bacterial stimulation 3) to examine the role of the MAPK and NFκB signalling pathways in the response of naïve and polarised macrophages to bacterial infection. Results: (i) polarisation of macrophages to an M1 state resulted in a higher secretion of pro-inflammatory cytokines (IL-6, IL12p70 and TNF-.). (ii) following bacterial stimulation M1 polarised macrophages had reduced pro-inflammatory cytokine release. (iii) M1 polarised macrophages have reduced MAPK and NFκB signalling as detected by western blot analysis. Conclusion: Following bacterial stimulation M1 polarised macrophages had reduced pro-inflammatory cytokine release which may in part be due to reduced MAPK and NFκB signalling. This data suggests that M1 polarisation states may play important roles in an endotoxin tolerant phenomenon in acute bacterial sepsis.Item Investigation of orally administrable bacteria for cancer therapy(University College Cork, 2021-01-06) Hogan, Glenn; Tangney, MarkDiminished progress in improving prognoses and treatment outcomes of several cancers, using conventional drugs, is being countered by investigations into a range of new, advanced formulations, of which bacteria-based anticancer therapies are one. Bacteria-based treatments hold an especially intriguing place within this group of disruptive medical technologies for the well-documented ability of bacteria to selectively colonise tumours following IV administration and replicate therein. Based on this property, bacteria have been studied both clinically and preclinically to evaluate their potential to promote tumour regression and confine noxious effects to the malignant tissue. Some of these studies have attempted to engineer the bacteria genetically to reduce their virulence and/or accentuate their capability to exert tumour-selective damage, but efforts to date have failed to penetrate clinical barriers and produce a safe, effective, and marketable bacteria-based product for cancer therapy. This thesis examines ways in which bacteria that are truly optimised for cancer treatment might be acquired and/or developed, using data derived from patient (Chapters 2, 3, and 4) and preclinical studies (Chapters 3, 4, and 5). The thesis can be divided into three main hypotheses: i) if naturally occurring bacteria can be identified in human tumour tissues, then these may be better tumour-targeting agents than the laboratory and probiotic strains that are routinely used in tumour-targeting studies (Chapters 2 and 3); ii) if bacteria that are administered orally to cancer patients can successfully colonise their tumours, then these may be safer and equally effective alternatives to intravenously administered microbes (Chapter 4); and iii) if bacteria are continually recycled through tumours via IV administration, then this may result in strains with improved tumour selectivity over their parental counterparts (Chapter 5). A prevailing concept throughout this work is that exposure to tumours, either via purposeful human intervention or natural colonisation, and the resultant evolutionary effects could adapt bacteria to the intratumoural environment more effectively than what genetic engineering would allow. This thesis contributes considerably to several bodies of knowledge, including the human tumour microbiota and the feasibility of administrable bacteria as vehicles for tumour-selective transmission of therapeutics. Chapters 2 and 3 use sequencing and culture techniques to develop an accurate and comprehensive picture of the human breast tumour microbiota. A culture-based assay was able to define the microbial diversity and community structure of breast tumours, when placed in the context of refined bioinformatic analyses. Despite a lack of evidence for viable, endogenous, tumour-selective bacteria, breast tumours contained a characteristic microbiological signature, as determined by deep sequencing, that warrants further exploration in varying tumour types to establish the diagnostic potential of this finding. Chapter 4 reports clinical data in relation to the translocation of probiotic bacteria from the GIT to distal tumours, using both FFPE specimens and fresh tissues. Results of this study, and corresponding animal experiments, shed light on the viability, or lack thereof, of this route of administration for prospective bacterial tumour-targeting studies in the clinic. Lastly, Chapter 5 is an account of the effects of tumour-to-tumour cycling on probiotic bacterial strains. The data in this chapter indicate that recycling bacteria through murine tumours can alter intratumoural growth dynamics and metabolic capacity, which was reflected in enhanced tumour persistence and biofilm formation. This work emphasises tumour recycling as a method by which bacteria can be adapted to the complex, heterogenous environment of cancerous tissue in a relatively simplistic way, which may have implications for the success of this treatment modality as it gains more clinical traction.