Exploring the effect of peri-operative inflammation on systemic and local breast cancer biomarkers
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Date
2024
Authors
Cullinane, Carolyn
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Background
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.
Description
Keywords
Peri-operative , Inflammation , Biomarkers , Breast cancer , Circulating tumour DNA
Citation
Cullinane, C. M. 2024. Exploring the effect of peri-operative inflammation on systemic and local breast cancer biomarkers. MD Thesis, University College Cork.