Cork Breast Research Group - 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 Surguvant inflammation and metabolism in colorectal surgery(University College Cork, 2020) Fleming, Christina A.; Redmond, Henry Paul; Corrigan, Mark; European Association for Cancer Research; Cork University HospitalBackground: It is believed that dysregulated perioperative inflammation (POI) and altered cellular and systemic metabolism in the perioperative period may predispose to colon cancer recurrence. This thesis examined the ability to harness perioperative body composition, cell free DNA (cfDNA) and metabolomic data and apply these as prognostic markers and potential therapeutic targets in the treatment of colon cancer. Hypothesis: Perioperative non-invasive methods of predicting clinical and cancer outcomes may be adopted in colon cancer, in particular anthropometric measurement, cfDNA analysis and profiling of the systemic metabolome. Aims: The aims of this thesis were as follows: 1. Analyse the association between CT-generated body composition parameters (BCPs), inflammation and colon cancer outcomes 2. Novel identification of the perioperative dynamics of cfDNA in colon cancer 3. Explore the perioperative metabolome in colorectal surgery 4. Investigate the effect of ‘surguvant’ systemic anti-inflammatory therapy on the perioperative metabolome in colorectal cancer surgery Methods: The association between CT-generated BCPs and five-year cancer recurrence and disease-specific mortality (DSM) were analysed using Mantel Cox log-rank test and Kaplan Meier curves generated. Inflammatory mediators driving these associations were characterised from pre-operative serum samples. Perioperative circulating cfDNA levels were measured at seven different times points (pre-operative and postoperative at 3hours, 6hours, 24hours, 48hours, POD3 and POD5). CEA levels were measured on the same patients for two years post-operatively. Comparison in trend (D) as defined by logistic regression was compared for perioperative cfDNA and post-operative CEA for identifying early colon cancer recurrence. The perioperative metabolome was characterised in patients undergoing both benign and cancer related colorectal surgery from serum samples (pre-operative, POD1 and POD5) using a Bligh-Dyer technique and GC-mass spectrometry. Metabolomic findings were explored to identify biomarker potential for predicting 30-day morbidity and five-year cancer outcomes (recurrence and DSM). The impact of body composition and systemic perioperative systemic anti-inflammatory therapy on the metabolome was also explored. Statistical analysis was performed using SPSS (v.26) and metabolomic analysis using MetaboAnalyst (v.5). Results: Both low skeletal muscle area (SMA) and high visceral to total fat ration (V:TFR) were associated with increased post-operative infectious complications [low SMA, OR 2.13 (95% CI 0.849-5.359)p=0.044; high V:TFR, OR 3.20 (95% CI 0.945-10.840)p=0.011] and five-year cancer recurrence [low SMA, HR 2.30 (95% CI 0.412-2.891)p=0.043; high V:TFR, HR 5.781(95% CI 0.662-9.450) p=0.024]. High V:TFR was also associated with five-year DSM [HR 5.916(95% CI 0.438-7.999)p=0.017]. Patients with low SMA who developed a cancer recurrence had higher circulating CRP(p=0.003), IL-6(p=0.004), VEGF(p=0.007) and CD14(p=0.029) and lower albumin(p=0.010), IL-2(p<0.001), IL-10 (p=0.004) and IFNg(p=0.018) levels. Those with high V:TFR who developed recurrence had higher levels of IL-6(p=0.030) and TNFa(p=0.029). Perioperative cfDNA was significantly associated with early cancer recurrence from 48Hrs (p=0.012) post-operatively [POD3 (p=0.01), POD5 (p=0.001)]. For identifying early recurrence, DcfDNA in the immediate perioperative period had a specificity of 97% (95%CI 86.51-99.87%) and 77.5% sensitivity (95%CI 62.5-87.7%). DCEA measured over the first two years post-operatively had similar accuracy 88.9% specificity (95%CI 56.5-99.4%) and 76.5% sensitivity (95%CI 63.24-86%) although valuable DCEA changes only occurred when recurrent disease was macroscopically evident. A differential perioperative metabolome was identified in benign (B-CRS) compared to colorectal cancer surgery (C-CRS). The urea cycle was most active in B-CRS and glycine and serine metabolism most active in C-CRS and in both cases followed by glutamate metabolism and ammonia recycling. Higher levels of ribose-5-phosphate (p=0.001), alanine (p=0.02), serine (p=0.01) and lactic acid (p<0.001) were observed throughout the perioperative period in patients with high V:TFR and higher succinic acid (p=0.03) in patients with low SMA. In stage II/III colon cancer through combined analysis of six specific metabolite concentrations (serine, proline, threonine, pyruvic acid, tryptophan, butanoic acid) a robust biomarker model for five year cancer recurrence was created [AUC 0.855 (95%CI 0.585-0.97) p<0.01]. Perioperatively, systemic anti-inflammatory therapy altered the measured metabolome. Conclusion: Body composition, cfDNA dynamics and metabolome alterations associated with poor prognosis in colon cancer may be identified in the perioperative period and could provide prognostic information for adjuvant therapy and surveillance decisionmaking. Metabolomic data in particular could further identify a potential for therapeutic targeting.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.