Return to Activities of Daily Living after Breast Cancer Surgery: An Observational Prospective Questionnaire-Based Study of Patients Undergoing Mastectomy with or without Immediate Reconstruction.

Background: Patients often ask about the time taken to return to activities of daily living (ADLs) after breast surgery, but there is a lack of data to give accurate guidance. We aimed to assess the feasibility of a study to determine the time taken to return to ADLs after mastectomy with or without breast reconstruction. Materials and Methods: A prospective multicentre, self-reported questionnaire-based feasibility study of women who had undergone mastectomy ± reconstruction was performed, between Jan 2017 and Dec 2019. Women were asked to self-report when they returned to 15 ADLs with a 5-option time scale for "return to activity." Results: The questionnaire was returned by 42 patients (median [range] age: 64 [31-84]). Of these, 22 had simple mastectomy, seven mastectomy and implant reconstruction, seven mastectomy and autologous reconstruction (DIEP), and six did not specify. Overall, over 90% could manage stairs and brush hair by two weeks and 84% could get in and out of the bath by four weeks. By 1-2 months, 92% could do their own shopping and 86% could drive. 68% of women employed returned to work within four months. Compared to simple mastectomy, patients undergoing reconstruction took a longer time to return to getting in/out of bath (<2 vs. 2-4 weeks), vacuuming (2-4 weeks vs. 1-2 months), and fitness (1-2 vs. 3-4 months). There was a slower return to shopping (1-2 months vs. 2-4 weeks), driving and work (both 3-4 vs. 1-2 months), and sports (3-4 vs. 1-2 months) in autologous reconstruction compared to implant reconstruction. Conclusion: This study is feasible. It highlights slower return to specific activities (particularly strength-based) in reconstruction patients, slower in autologous compared with implant reconstruction. The impact on return to ADLs should be discussed as part of the preoperative counselling as it will inform patients and help guide their decision making. A larger study is required to confirm these results.

Patient reported outcome measures in a cohort of patients at high risk of breast cancer treated by bilateral risk reducing mastectomy and breast reconstruction.

BACKGROUND: Many women with increased lifetime risk of developing breast cancer, due to pathogenic gene variants or family history, choose to undergo bilateral risk reducing mastectomies (BRRM). Patient reported outcome measures (PROMS) are an increasingly important part of informed consent but are little studied in women undergoing BRRM. METHODS: We used a validated PROMS tool for breast reconstruction (BREAST-Q) in 297 women who had BRRM and breast reconstruction, 81% of whom had no malignancy (Benign Group, BG) and 19% in whom a perioperative breast cancer was diagnosed (Cancer Group, CG). 128 women also completed a Hospital Anxiety & Depression Score (HADS) questionnaire to test if preoperative HADS score could predict PROMS outcomes. RESULTS: Women in the CG had lower PROMS scores for satisfaction with their breasts, nipple reconstruction and sexual wellbeing. Both groups reported equal satisfaction with BRRM outcome and psychosocial well-being. Physical well-being PROMS of the abdomen and chest were high in women in both groups as were scores for satisfaction with the care they received. The CG group reported suboptimal quality of patient information. A higher presurgical HADS anxiety score predicted less favourable postoperative psychosocial well-being despite similar levels of satisfaction with aesthetic outcome. CONCLUSION: We show a high degree of patient reported satisfaction by woman undergoing BRRM and reconstruction. There was a negative association with a cancer diagnosis on quality of life PROMS and higher preoperative anxiety levels negatively affected postoperative psychosocial well-being. These important findings should be part of the informed consent process during preoperative counselling.

Innovations for the future of breast surgery.

BACKGROUND: Future innovations in science and technology with an impact on multimodal breast cancer management from a surgical perspective are discussed in this narrative review. The work was undertaken in response to the Commission on the Future of Surgery project initiated by the Royal College of Surgeons of England. METHODS: Expert opinion was sought around themes of surgical de-escalation, reduction in treatment morbidities, and improving the accuracy of breast-conserving surgery in terms of margin status. There was emphasis on how the primacy of surgical excision in an era of oncoplastic and reconstructive surgery is increasingly being challenged, with more effective systemic therapies that target residual disease burden, and permit response-adapted approaches to both breast and axillary surgery. RESULTS: Technologies for intraoperative margin assessment can potentially half re-excision rates after breast-conserving surgery, and sentinel lymph node biopsy will become a therapeutic procedure for many patients with node-positive disease treated either with surgery or chemotherapy as the primary modality. Genomic profiling of tumours can aid in the selection of patients for neoadjuvant and adjuvant therapies as well as prevention strategies. Molecular subtypes are predictive of response to induction therapies and reductive approaches to surgery in the breast or axilla. CONCLUSION: Treatments are increasingly being tailored and based on improved understanding of tumour biology and relevant biomarkers to determine absolute benefit and permit delivery of cost-effective healthcare. Patient involvement is crucial for breast cancer studies to ensure relevance and outcome measures that are objective, meaningful, and patient-centred.

This article describes how future innovations in science and technology influence the management of breast cancer from a surgical perspective. This work was undertaken in response to the Commission on the Future of Surgery project initiated by the Royal College of Surgeons of England.

Risk factors for complications and implant loss after prepectoral implant-based immediate breast reconstruction: medium-term outcomes in a prospective cohort.

BACKGROUND: Prepectoral implant-based breast reconstruction with acellular dermal matrix has become an increasingly popular option for selected patients. There are no randomized data to demonstrate short- or long-term outcomes. Cohort studies to date have demonstrated safety, but risk factors for complications are unknown. METHODS: A prospective cohort study of all patients undergoing prepectoral implant-based breast reconstruction between 2013 and 2019. Clinical factors and those related to reconstruction were analysed in relation to complications and implant loss using univariable and multivariable logistic regression. RESULTS: A total of 469 reconstructions were undertaken in 289 women; the majority of reconstructions were performed using a one-stage direct-to-implant technique with acellular dermal matrix. Median follow-up was 21 (range 2-71) months. Minor complications were seen after 11·2 per cent of reconstructions, major complications after 5·9 per cent, and the rate of implant loss by 3 months was 3·1 per cent. In the final multivariable model, sentinel node biopsy (odds ratio (OR) 5·06, 95 per cent c.i. 2·00 to 12·80), axillary clearance (OR 6·67, 1·17 to 37·94) and adjuvant radiotherapy (OR 7·11, 1·60 to 31·61) were independent risk factors for development of a major complication, and sentinel node biopsy (OR 4·32, 1·23 to 15·22) for implant loss. CONCLUSION: Prepectoral implant-based breast reconstruction has acceptable medium-term results but careful patient selection is advised.

Does tamoxifen have a therapeutic role outside of breast cancer? A systematic review of the evidence.

INTRODUCTION: Tamoxifen is a widely used hormonal based therapy for breast cancer in the adjuvant and metastatic setting, prolonging overall and recurrence-free survival. There has been increasing interest in the potential for novel "off-target" effects of tamoxifen and its metabolite N-desmethyltamoxifen across a number of cancer types. We aim to review the current literature regarding the potential use of tamoxifen in other primary malignancies. METHOD: A qualitative systematic review was performed according to the PRISMA guidelines using pre-set search criteria across the PubMed, Cochrane and Scopus databases from 1985 to 2019. Additional results were generated from included papers references. RESULTS: A total of 324 papers were identified, of which 47 were included; a further 29 articles were obtained from additional referencing to give a total of 76 articles. Clinical trials have demonstrated benefits with the use of tamoxifen in isolation and combination, specifically in patients with advanced non-resectable malignancy, however results are not consistent across the literature. In vivo data consistently suggests that off target effects of tamoxifen are mediated through the ceramide pathway or through inhibition of protein kinase C (PKC). CONCLUSIONS: With increased focus upon the potential of repurposing drugs, tamoxifen may be a candidate for repurposing in the wider cancer setting. There is evidence to suggest that the ceramide or PKC pathway could act as a therapeutic target for tamoxifen or alternative chemotherapeutics and merits further investigation.

Circulating tumour cells and hypercoagulability: a lethal relationship in metastatic breast cancer.

PURPOSE: Circulating tumour cells (CTCs) are a marker of poor prognosis and are associated with increased risk of venous thromboembolism in metastatic breast cancer (MBC). We aimed to determine if the presence of CTCs and plasma markers of hypercoagulability [thrombin-antithrombin III (TAT), fibrinogen and D-dimer] are biomarkers of survival in MBC. METHODS/PATIENTS: In a prospective study of MBC patients, CTC (CellSearch®) enumeration and plasma TAT, fibrinogen and D-dimer measured prior to commencement of treatment for disease progression were correlated to overall survival. RESULTS: At study completion, of 50 MBC patients recruited (median age 59 years, range 36-82), 40 patients had died (median survival 417 days, range 58-2141). CTCs (≥ 1/7.5 ml) were identified in 16 patients (median number of cells per 7.5 ml, 3 (range 1-31) and were associated with systemic hypercoagulability (medians TAT: 8.1 vs. 5.2 ng/ml, p = 0.03; fibrinogen: 4.3 vs. 3.1 g/l, p = 0.03; D-dimer: 1327 vs. 683 ng/ml, p = 0.0001). At 1 year, of 16 patients with ≥ 1 CTC, 7 had died (44%), compared to 5 of 26 (19%) patients in the no-CTC group. The presence of ≥ 1 CTC was associated with a trend for reduced overall survival (median 455 days vs. 614 days, p = 0.15). Plasma TAT inversely correlated with survival and was significantly higher in patients dying within 1 year (median 9.8 vs. 5.2 ng/ml, p = 0.004) whilst D-dimer showed a trend for reduced 1-year survival (median 1211 vs. 817 ng/ml, p = 0.06). MBC patients with combined high D-dimer (≥ 895 ng/ml) and CTC positivity (≥ 1/7.5 ml whole peripheral blood) had significantly reduced survival (p = 0.04). CONCLUSIONS: The correlation between CTCs, hypercoagulability and reduced survival in MBC suggests the coagulation system supports tumour cell metastasis and is, therefore, a potential therapeutic target.

Colorectal cancer and thrombosis.

SIGNIFICANCE: Colorectal cancer (CRC), results in a hypercoagulable state which manifests clinically as venous thromboembolism (VTE), often presenting as a deep vein thrombosis (DVT) or pulmonary embolism (PE). The consequences of VTE in CRC can be devastating, resulting in long-term morbidity and are a frequent cause of death, even amongst those who would have otherwise had a favourable cancer prognosis. The incidence of VTE in all cancers is increasing, whilst the exact incidence of VTE in CRC is likely to be underestimated. All cancer treatments increase the risk of VTE in an already at risk population. CRITICAL ISSUES: CRC-associated VTE is a challenging entity to manage with recurrences occurring more frequently in cancer patients, despite anticoagulation. Anticoagulation, whether treatment or prophylactic, increases the risk of bleeding, especially in patients with cancer. Although strong evidence underpins the initial management of cancer-associated VTE, there is uncertainty with regard optimum treatment duration. For VTE prevention, extended (28 days), pharmacological thromboprophylaxis post CRC surgery is internationally recommended. Pharmacological thromboprophylaxis is not routinely recommended for nonhospitalised patients receiving chemotherapy. FUTURE DIRECTIONS: There is growing evidence of a symbiotic relationship between cancer biology and the clotting system. Tissue factor (TF), the initiator of the clotting pathway, promotes cancer via clotting dependent and independent mechanisms. Clotting pathway factors, including TF, may have utility as biomarkers in CRC, for assessment of VTE risk in addition to cancer prognosis. The clotting system may also be a target for potential anti-cancer therapies, either via existing anticoagulants or experimental direct TF inhibitors.

OC-09 - Microparticle-mediated transfer of TF hypercoagulability between cancer cells.

INTRODUCTION: Tissue Factor (TF) promotes apoptosis-resistance and facilitates haematogenous metastasis. Cancer cells release TF-bearing microparticles (TF-MP) which have pro-coagulant activity. TF-MPs from cancer cells induce increase in TF expression and procoagulant activity of recipient endothelial cells. Drug resistance microparticles transfer between cancer cells, resulting in induction of recipient cell drug resistance, however transfer of TF-MPs between cancer cells has not been demonstrated. AIM: To determine if microparticles isolated from TF expressing cancer cell lines can induced TF-mediated procoagulant activity in cell lines that do not express TF. MATERIALS AND METHODS: A7 cells (a melanoma cell line that does not express TF) were transformed to express TF with adenoviral transformation. In addition, the TF-expressing breast cancer cell line MDAMB231 was used. Media was collected from (1) wild-type A7 (A7-WT) cells, which acted as control (2) MDAMB231 cells, (3) A7 cells transformed with 'low' levels of TF-adenovirus and (4) A7 cells transformed with 'high' levels of TF-adenovirus. MPs were isolated from media by centrifugation using standard techniques. Isolated MPs were incubated with wild-type A7 (A7-WT) cells overnight. Pro-coagulant activity of A7-WT cells was assessed using a FXa generation assay. RESULTS: i) A7-WT cells incubated overnight with MDAMB231-derived MPs produced an almost 5 fold increase in procoagulant activity compared to control (Mean 3.5 mOD/min S.E 0.32 vs control 0.68 S.E. 0.05, p<0.01). ii) MPs released from A7-TF cells induced a dose-dependant increase in cell based procoagulant activity when incubated with A7-WTs compared to control:A7-WT cells incubated with control MPs (Control): mean mOD 0.83, S.E. 0.07A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 1.27, S.E. 0.06 (p=0.04 vs control)A7-WT cells incubated with A7-TF derived MPs (low levels of TF-adenovirus): mean mOD 2.77, S.E. 0.23 (p<0.01 vs control). CONCLUSIONS: These results demonstrate, for the first time, microparticle-mediated transfer of TF procoagulant activity between cancer cells. MPs shed by cancer cells in vivo have been implicated in cancer cell progression and hypercoagulability. The acquisition and spread of of MP-mediated TF activity has implications for cancer cell biology and cancer-induced hypercoagulability.

PO-09 - Incidence of pre-operative and post-operative deep vein thrombosis in colorectal cancer surgery. Interim results of a prospective clinical study.

INTRODUCTION: Venous thromboembolism (VTE) is a major cause of death in patients undergoing colorectal cancer surgery and usually arise from Deep Vein Thrombosis's (DVTs). In patients with cancer below knee and asymptomatic DVTs are at risk of propagating and result in a VTE. Retrospective, population based studies predating extended course venous thromboprophylaxis report an incidence of symptomatic VTE in colorectal cancer patients of approximately 5.5%. Clinical studies have suggested that pre-operative d-dimer may predict the development of post-operative DVTs. AIM: We undertook a multicentre prospective study to screen colorectal cancer patients pre- and post-operatively for the presence of DVTs. The aim of the study was to determine the incidence of DVTs in patients undergoing elective, curative surgery for colorectal cancer. The study also aimed to identify histological and clinical factors that pre-dispose to development of a DVT. The role of pre-operative d-dimer in predicting the development of post-operative DVTs was also analysed. MATERIALS AND METHODS: Patients at four hospitals undergoing elective curative surgery for colorectal cancer were recruited pre-operatively. Exclusion criteria were: previous VTE, previous malignancy, anti-coagulants. Bilateral full leg venous duplex was performed pre-operatively and at six weeks post-surgery. Plasma D-dimer was measured pre-operatively. RESULTS: Of 60 patients undergoing pre-operative duplex, five (8.3%) had below knee, asymptomatic DVTs. These patients were then excluded from further analysis. Of the remaining 55, 48 had post-operative duplex examination. Three (6.3%) developed post-operative DVTs all of which were asymptomatic and below knee. There were no symptomatic DVTs. Two of these patients had received extended course venous thromboprophylaxis and the other received inpatient thromboprophylaxis. Development of post op DVT was associated with lymph node positivity (p=0.02). There were no other histological, surgical or demographical factors that predicted the development of post-operative DVTs. Pre-operative D-dimer was higher in patients who developed a post-operative DVT compared to those who did not [1,275 ng/L (95%CI: 780 - 1770 ng/L) vs 805 ng/L (95% CI: 632 - 980 ng/L) p=0.03]. CONCLUSIONS: Of patients diagnosed with operable colorectal cancer, 8% have pre-operative asymptomatic DVT, and a further 6% develop DVT despite thromboprophylaxis. The subgroup of patients with lymph node involvement may benefit from more aggressive anticoagulation as they are at increased risk of DVT despite extended course anticoagulation. Pre-operative D-dimer may offer a predictive method to identify patients at risk of post-operative DVT.

PO-11 - Thrombin and cancer stem-like cells: in vitro support for breast cancer anticoagulation.

INTRODUCTION: Cancer stem-like cells (CSCs) are tumour initiating, resistant to chemotherapy and play a role in metastasis. We have previously shown thrombin pathway signalling regulates CSC activity: thrombin pathway activation with FVIIa or through TF overexpression increases CSC activity whilst TF siRNA knockdown decreases it. We hypothesise that NOACs, by targeting specific factors in the thrombin pathway, may inhibit CSC activity. AIM: In breast cancer cell lines, to determine the effect of i) thrombin and ii) dabigatran (Boehringer Ingelheim), a direct thrombin inhibitor on cancer stem cell activity. MATERIALS AND METHODS: MDA-MB-231, MCF-7, SKBR3 and MDA-MB-157 cell lines representing the spectrum of breast cancer subtypes were cultured with: i) 0.1 NIH Units/ml human thrombin (Sigma) ii) 0.5µM Dabigatran (clinically relevant plasma concentration). Mammosphere culture is an established in vitro assay to measure CSC activity. Mammosphere forming efficiency (MFE) was calculated from the proportion of plated cells forming mammospheres in non-adherent culture, (each experiment, n=≥4, two-tailed independent t-test). RESULTS: Thrombin increased MFE in the high PAR-1 expressing MDA-MB-231 and MDA-MB-157 cell lines, but not the low PAR-1 expressing MCF-7/SKBR3 cell lines as compared to untreated controls (MDA-MB-231 mean (range): thrombin treated: 0.91 (0.70-1.11) vs control: 0.73 (0.51-0.93)%, p=<0.04; MDA-MB-157: thrombin treated: 0.58 (0.45-0.69) vs control: 0.37 (0.31-0.47)%, p=<0.001). Dabigatran abrogated the stimulatory effect of thrombin on MFE in thrombin-treated MDA-MB-231 and MDA-MB-157 cells (MDA-MB-231 mean (range) thrombin+Dabigatran: 1.10 (0.83-1.41) vs thrombin only: 1.45 (1.32-1.66)%, p=<0.01); MDA-MB-157 thrombin+Dabigatran: 0.48 (0.39-0.56) vs thrombin only: 0.58 (0.45-0.69)%, p=<0.05). CONCLUSIONS: The stimulation of mammosphere formation by thrombin and reduction by thrombin inhibitor treatment indicates a functional relationship between cancer stem-like cells and coagulation. This suggests possible clinical utility of novel oral anticoagulants in targeting this critical cancer cell subpopulation.

PO-21 - Stromal fibroblasts in preinvasive breast cancer (ductal carcinoma in situ, DCIS) demonstrate a cancer-like procoagulant phenotypic switch that may facilitate invasion.

INTRODUCTION: Ductal carcinoma in-situ (DCIS) is a preinvasive breast cancer where cancer cells remain confined within the ductal basement membrane. However, genotypic changes have been identified in stroma surrounding DCIS, outside the basement membrane. Stromal fibroblasts undergo phenotypic change in cancer to promote tumour angiogenesis, proliferation, immunosuppression and metastasis and in vivo can induce invasion of DCIS. Phenotypic changes in DCIS stromal fibroblasts may potentially act as a precursor for invasion. AIM: To determine if stromal fibroblasts in DCIS have procoagulant changes similar to those seen in cancer-associated fibroblasts in invasive breast cancer. MATERIALS AND METHODS: As part of the prospective cohort study CHAMPion (Cancer induced Hypercoagulabulity as a Marker of Prognosis), patients with DCIS (n=72) and invasive breast cancer (n=292) were recruited. Stromal fibroblasts in tumour and corresponding normal breast tissue (distant from the cancer) were quantified (percentage IHC stained) for tissue factor (TF), thrombin, PAR1 and PAR2. Fibroblasts were identified morphologically, at a minimum distance of 0.2mm from ductal tissue, to avoid myoepithelial scoring. Scoring was performed in duplicate by two independent pathologists. RESULTS: Fibroblast TF expression was present in normal breast tissue (mean 43% ([SD 27%]) but markedly increased in DCIS (mean 62% [SD 27%], p=0.002). Fibroblast TF expression was further increased in invasive breast cancer (mean 74% [SD 23%], normal vs invasion, p<0.001; DCIS vs invasion, p=0.03). Fibroblast thrombin and PAR2, but not PAR1, expression was increased in DCIS compared to normal (thrombin: 60% vs 42%, p<0.001; PAR2: 58% vs 41%, p=0.002), however no further significant increase was seen in invasive cancer (thrombin 63%, PAR2 61%). Fibroblast tissue factor correlated with fibroblast thrombin expression (p<0.001, r=0.4) and fibroblast PAR2 expression (p<0.001, r=0.5), with thrombin and PAR2 expression also correlating (p<0.001, r=0.4). CONCLUSIONS: Procoagulant phenotypic changes, in terms of increased TF, thrombin and PAR2 expression, occur in stromal fibroblasts at the preinvasive stage. It needs to be determined if this change is functional and therefore a potential therapeutic target for preventing transition to invasion.

PO-32 - Patient, tumour and operative factors influencing perioperative hypercoagulability in colorectal cancer.

INTRODUCTION: Up to 6% of patients develop venous thromboembolism (VTE) following elective colorectal cancer surgery despite thromboprophylaxis. Clinical practices for perioperative thromboprophylaxis remains variable, particularly the use and duration of extended thromboprophylaxis. Identification of factors associated with a prolonged postoperative hypercoagulable state may allow the development of algorithms that allow more targeted thromboprophylaxis. AIM: To identify patient, tumour and surgical risk factors for prolonged (two and six weeks) hypercoagulability in colorectal cancer patients undergoing surgical resection. MATERIALS AND METHODS: In a prospective cohort study (Cancer-induced Hypercoagulability As a Marker of Prognosis [CHAMPion]), plasma d-dimer was measured at 2 and 6weeks post-operatively in patients undergoing elective, curative resection for colorectal cancer. Hypercoagulability (raised D-dimer) at 2 and 6weeks was correlated with patient, tumour and operative factors. RESULTS: Of the 62 patients recruited (median age 69years [range 39-90]), 37 were male. D-dimer was increased in females compared to males at six weeks (geometric mean (GM) 1,287ng/ml [95% CI 944 - 1,755 vs. 821ng/ml (95% CI 633 - 1064) p=0.03]. Age, smoking, hypertension, use of antiplatelet medication, BMI and WHO performance status were not associated with a prolonged hypercoagulable state. There were no tumour factors (including size/T stage/lymph node involvement/differentiation) associated with a prolonged hypercoagulable state. D-dimer was increased in patients undergoing open surgery (n=39) compared to laparoscopic surgery (n=23) at 2weeks (GM 2,337ng/ml [95% CI 1,806-3,023] vs. 1,212ng/ml [95% CI 898-1,629], p=0.001) and 6weeks (GM 1,162ng/ml [95% CI 818-1647] vs. 723ng/ml [95% CI 533-982]p=0.04). Operative time was not associated with prolonged hypercoagulability. D-dimer had a trend to be increased at 2weeks in patients receiving perioperative blood transfusions (n=8) compared to those that did not (n=54) (GM 2,618ng/ml [95% CI 1,525-4,536] vs. 1613ng/ml [95% CI 1,236 - 2,100] p=0.08). CONCLUSIONS: Even in this relatively small cohort of patients female gender, open surgery and receiving a blood transfusion are associated with on-going hypercoagulability up to six weeks post operation. This may represent a group where thromboprophylaxis should be targeted.

PO-31 - Circulating tumour cells and hypercoagulability: a lethal relationship in metastatic breast cancer.

INTRODUCTION: Circulating tumour cells (CTCs) are a marker of poor prognosis and are associated with increased risk of venous thromboembolism in metastatic breast cancer. AIM: We aimed to correlate presence of CTCs and markers of hyper-coagulability (D-dimer, fibrinogen and thrombin-antithrombin [TAT]) with survival in metastatic breast cancer. MATERIALS AND METHODS: In a prospective study, enumeration of CTCs (CellSearch) and D-dimer, fibrinogen and TAT (ELISA) were measured at a single timepoint in 50 MBC (median age 59, range 36-82) patients undergoing active treatment. Survival data was determined at a median follow-up of 366days (range 58-986). RESULTS: To date, 25 patients have died (median survival 566days, range 135-978). CTCs (>1/7.5ml) were identified in 13 patients (range 2-31) and were associated with increased markers of hypercoagulability [D-dimer: median 1814 (IQR 2700) vs 755 (IQR 735) ng/ml, p=0.004; fibrinogen: median 4.2 (IQR 1.9) vs 3.2 (1.3) g/l, p=0.05; TAT: median 6.2(IQR 6.3) vs 4.7 (5.2) ng/ml, p=0.1]. CTCs were associated with visceral compared to just bony metastases (p=0.03) and their presence was associated with a trend for reduced survival (295days (CI: 0-652) vs 737days (CI: 186-1288), p=0.1). There was no correlation between CTCs /markers of hypercoagulability and age, oestrogen receptor, progesterone receptor or Her2 status. D-dimer, fibrinogen and TAT all inversely correlated with survival and were all significantly higher in patients dying within 1year (D-dimer: 1098 (IQR 1122) vs 723 (IQR 735) ng/ml, p=0.03; fibrinogen: 4.4 (1.1) vs 3.2 (0.8) g/l, p=0.004; TAT: 8.1 (6.3) vs 4.7(3.1) ng/ml, p=0.03 [analysis excludes patients with <1year follow-up, n=13]). D-dimer >1,500ng/ml was associated with significantly reduced survival (295days [CI: 0-615] vs 836days [404-1267], p=0.05). On Cox regression, D-dimer, but not fibrinogen or TAT was associated with an increased risk of death (HR 1.3 per 1,000ng/ml D-dimer, p=0.07). CONCLUSIONS: The correlation between CTCs, hypercoagulability and reduced survival in metastatic breast cancer suggests a possible role for the coagulation system in supporting tumour cell metastasis and is therefore a potential therapeutic target.

PO-36 - Thrombin Inhibition Preoperatively (TIP) in early breast cancer, the first clinical trial of NOACs as an anti-cancer agent: trial methodology.

INTRODUCTION: Breast cancer is associated with a 3-4 fold increased risk of VTE. These patients have a 4-fold lower survival than those remaining free of VTE, implying VTE is a surrogate marker for aggressive cancer. Tumour expression of thrombin pathway markers are increased in the oestrogen receptor negative (ER-), high Ki67, more aggressive breast cancer subtypes. In in vitro and in vivo studies, the thrombin pathway promotes cancer growth and metastases, highlighting the potential role of the thrombin pathway as a therapeutic target in cancer. AIM: To determine whether 14days of a preoperative oral Factor Xa inhibitor (Rivaroxaban) in oestrogen receptor negative early breast cancer patients results in inhibition of tumour proliferation as determined by a reduction in tumour Ki67 from baseline (pre-treatment) to 14days post treatment start (at time of surgical excision). RATIONALE: The TF-VIIa-FXa complex activates Protease Activated Receptor (PAR)2 to induce angiogensis, growth factors and tumour cell migration. Thrombin, in part via PAR1, induces angiogenesis, tumour cell proliferation as well as in vivo metastasis. In early breast cancer, TF and PAR2 expression is increased in the stroma, particularly in the more aggressive ER-, high Ki67 (proliferation) cancers. Rivaroxaban is an orally active direct Factor Xa inhibitor. Through inhibition of the TF-FVIIa-FXa complex, it can downregulate TF-FVIIa-FXa activation of PAR2, and inhibit conversion of prothrombin to thrombin. We hypothesise that 14days of Rivaroxaban will reduce breast cancer proliferation, as a surrogate marker for anti-cancer efficacy, in early breast cancer patients awaiting resectional surgery. RESULTS: Trial methodology: A multi-centre phase II preoperative 'Window-of Opportunity' randomised controlled trial of Rivaroxaban compared to no treatment in ER-, stage I-III early breast cancer patients. Patients will be randomised 1:1:1 (Rivaroxaban 20mg od: Rivaroxaban 10mg od: no treatment) and receive 14 (+/-3) days of treatment in the window between diagnosis and surgery. Randomisation will be blinded to pathologists, but not to patients or clinicians. Primary analysis will be based on the two Rivaroxaban arms being combined to form a Rivaroxaban: no treatment, 2:1 trial design, with change in Ki67 from baseline (pre) to post Rivaroxaban/no treatment (post) being the primary endpoint, and the no treatment arm acting as a reference group. Subgroup analysis of the Rivaroxaban arm (20mg od:10mg od) will allow assessment of dose-response. ACKNOWLEDGEMENT: Funder: National Institute for Health Research Eudract No: 2014-004909-33 REC Number: 15/NW/0406 UKCRN ID: 19731 Expected commencement: January 2016. For further information please contact Chief Investigator: cliona.kirwan@manchester.ac.uk.

PO-51 - Expression of proteins of the tissue factor thrombin pathway is upregulated in the stroma and epithelium of colorectal cancer.

INTRODUCTION: Colorectal cancer expression of Tissue Factor (TF), PAR1 and PAR2 is associated with a poor prognosis. Their stromal, rather than epithelial, expression has prognostic significance in other cancers, this has not been explored in colorectal cancer. AIM: We aimed to determine the expression patterns of Tissue Factor (TF), PAR1 and PAR2 and thrombin in colorectal cancer and normal tissue. MATERIALS AND METHODS: Cancer and distant normal tissue were sampled from 37 patients. Expression of TF, Thrombin, PAR1 and PAR2 were determined by immunohistochemistry. Two observers scored expression level (0-3) in individual cells. Percentage of cells having each level of expression was determined and an H-score calculated which are given with 95% CI. RESULTS: Normal epithelium did not express TF, but it was expressed by cancer epithelium (36.5 (95% CI 17.6 - 55.4) p<0.001). Thrombin expression was increased in cancer vs normal epithelium (126.2 (95% CI 110.6 - 141.7) vs 101.6 (95% CI 92.5-110.8) p=0.01) as was PAR2 (172.4 (95% CI 152.9-191.8) vs 123.4 (95% CI 107.8-139.0) p<0.001). The increase in cancer epithelium PAR1 expression compared to normal (105.4 (95%CI 84.3-126.5) vs 89.0 (95% CI 80.4-97.6)) was not significant. Normal stroma did not express TF or thrombin however both were expressed by cancer stroma (TF 46.3 (95% CI 24.6-68.0) p<0.001, thrombin 11.4 (95% CI 6.2-16.7) p<0.001). PAR1 and PAR2 were both expressed in normal stroma but demonstrated increased expression in cancer stroma (cancer vs normal; PAR 1: 130.7 (95% CI 112.2-149.2) vs 19.5 (95% CI 11.24-27.7) p<0.001; PAR2: 21.5 (95% CI 12.9-30.1) vs 2.21 (95% CI 0.49-3.92) p<0.001). CONCLUSIONS: Upregulated expression of tissue thrombin pathway proteins is seen in colorectal cancer in both epithelial and stromal cells. Procoagulant tumour cells and tumour microenvironment may provide a novel therapeutic target for treatment in colorecal cancer.

PO-52 - Effect of tissue factor expression by colorectal cancer on cancer stem cell activity.

INTRODUCTION: Abnormal Tissue factor (TF) expression occurs in many cancers including colorectal and is associated with a poor prognosis. TF expression promotes cancer stem cell (CSC) activity in breast and squamous cell carcinoma (SCC). AIM: The aim of the research is to determine the effect of TF expression by colorectal cancer cell lines on colorectal CSC activity. MATERIALS AND METHODS: Colorectal cancer cell lines with high (DLD-1) and low (SW620) TF expression were stably transduced to knock down (TF-ve) and over express (TF+ve) TF. CSC activity was assessed using the cancer sphere assay (sphere forming efficiency (SFE)=spheres formed / cells plated) and by ALDH1 expression (by florescent activated cell sorting). When comparing transduced cells to their control the ALDH1 expression was normalised to their control. RESULTS: The naturally high TF expressing cell line (DLD-1) compared to the low TF expressing cell line (SW620) had a reduced SFE (0.7 (SD 0.39) vs 1.38 (SD 0.68), p<0.05) and less cells expressed ALDH1 (15.3% (SD 1.2) vs 26.9% (SD 2.1), p=0.02). DLD-1 TF+ve compared to its control had a reduced SFE (0.33 (SD 0.14) vs 0.66 (SD 0.42), p<0.01) and less cells expressed ALDH1 (0.72 (SD 0.069) p=0.001) (normalised to negative control). There was no difference in SFE (1.21 (SD 0.56) vs 1.29 (SD 0.50) p=0.4) and ALDH1 expression (0.81 (SD 1.00) p=0.5) between SW620 TF+ve and its control. DLD-1 TF-ve and SW620 TF-ve had increased SFE compared to their controls (DLD-1: 0.63 (SD 0.27) vs 0.41 (SD 0.35) p<0.01; SW620: 2.03 (SD 0.86) vs 1.21 (SD 0.70) p<0.01), and increased ALDH expression (DLD-1: TF-ve 1.23 (SD 0.74)p=0.04 and SW620: TF-ve 1.31 (SD 0.08) p<0.001 normalised to controls). CONCLUSIONS: Unlike in breast and SCC, in colorectal cancer cell lines TF inhibits CSC activity. This has significance if anti-tissue factor treatments are considered as an anti-cancer treatment as this may increase the cancer stem cell activity.

PO-50 - The effect of tissue factor expression on colorectal cancer cell proliferation.

INTRODUCTION: Tissue factor (TF) is abnormally expressed in many cancers including colorectal and is associated with a poor cancer prognosis. Colorectal cancer cell lines expressing TF produce faster growing tumours. In lung cancer, TF inhibition has been shown to reduce proliferation AIM: We aimed to determine if TF expression and activity increases cellular proliferation in colorectal cancer cell lines. MATERIALS AND METHODS: DLD-1 and SW620 colorectal cell lines were transduced with cDNA to over express TF (TF+ve). Proliferation was determined by Alamar blue assay where level of absorption indicates the number of living cells, expressed as an arbitrary unit of absorption (u). Factor VIIa (TF ligand) at increasing concentrations was used to determine the effect of TF activity on proliferation. Downstream marker of TF activity (MAPK phosphorylation) was assessed by Western blot and correlated with proliferation. RESULTS: There was a significant increase in proliferation in both DLD-1 TF+ve and SW620 TF+ve compared to their negative controls at 42 hours (DLD1 TF+ve: 5,455u (SD 2,485u) vs 2,246u (SD 1,107u) p<0.001); SW620 TF+ve: 414u (SD 96u) vs 286u (SD 114u) p<0.05). When factor VIIa (FVIIa) was added in concentrations from 0nM to the supra-physiological concentration of 25nM there was a dose-dependent increase in proliferation up to physiological levels (0.1nM) which was further increased in the TF+ve cell lines. Fold change from baseline 0 vs 0.1nM FVIIa (DLD-1: 3.22u (SD 0.61u) vs 6.17u (SD 2.21u) p<0.05; SW620 2.33u (SD 2.21u) vs 4.69u (SD 0.61u) p<0.05). The increase in proliferation was reflected in the phosphorylation of MAPK which was increased by TF overexpression alone and further increased by FVIIa in a dose-dependent fashion. CONCLUSIONS: Increased TF expression and activation is associated with increased cellular proliferation. This effect appears to be exerted via MAPK pathways. Tissue factor may provide a therapeutic target in colorectal cancer.