Hudhaifah Shaker

Tissue Factor promotes breast cancer stem cell activity in vitro

Cancer stem cells (CSCs) are a subpopulation of cells that can self-renew and initiate tumours. The clotting-initiating protein Tissue Factor (TF) promotes metastasis and may be overexpressed in cancer cells with increased CSC activity. We sought to determine whether TF promotes breast CSC activity in vitro using human breast cancer cell lines. TF expression was compared in anoikis-resistant (CSC-enriched) and unselected cells. In cells sorted into of TF-expressing and TF-negative (FACS), and in cells transfected to knockdown TF (siRNA) and overexpress TF (cDNA), CSC activity was compared by (i) mammosphere forming efficiency (MFE) (ii) holoclone colony formation (Hc) and (iii) ALDH1 activity. TF expression was increased in anoikis-resistant and high ALDH1-activity T47D cells compared to unselected cells. FACS sorted TF-expressing T47Ds and TF-overexpressing MCF7s had increased CSC activity compared to TF-low cells. TF siRNA cells (MDAMB231, T47D) had reduced CSC activity compared to control cells. FVIIa increased MFE and ALDH1 in a dose-dependent manner (MDAMB231, T47D). The effects of FVIIa on MFE were abrogated by TF siRNA (T47D). Breast CSCs (in vitro) demonstrate increased activity when selected for high TF expression, when induced to overexpress TF, and when stimulated (with FVIIa). Targeting the TF pathway in vivo may abrogate CSC activity.

Abstract P6-08-30: The thrombin clotting pathway is upregulated in the stroma of pre-invasive breast cancer and further upregulated in aggressive invasive breast cancer phenotypes

BACKGROUND Components of the thrombin (extrinsic) clotting pathway are upregulated in cancer. The clotting pathway factors tissue factor (TF) and Thrombin promote tumour progression through protease activated receptors PAR2 and PAR1 respectively. AIMS To determine if tumour expression (epithelial and stromal) of a procoagulant phenotype is associated with aggressive breast cancer phenotypes and reduced survival. METHODS Tumour expression of TF, thrombin, PAR1 and PAR2 was determined by immunohistochemistry in two cohorts. PROSPECTIVE STUDY Early invasive breast cancer (n=199), ductal carcinoma in situ (DCIS, n=42) and normal breast tissue samples (n=121). RETROSPECTIVE STUDY Early invasive breast cancer patients (n=144) with median follow-up of 69 (range 4 to 91) months. Procoagulant phenotype expression was correlated with tumour grade, proliferation (Ki67), ER and HER2 status (both cohorts), survival and recurrence (retrospective cohort). RESULTS PROSPECTIVE STUDY Epithelium Thrombin (p Stroma TF, Thrombin, PAR1 and PAR2 were increased in the stroma of DCIS compared to normal breast stroma (p In invasive breast cancer, TF was increased in invasive cancer compared to DCIS and compared to normal breast tissue (p TF, thrombin, PAR1 and PAR2 were increased in high proliferating (p RETROSPECTIVE STUDY Stroma As with the prospective study, thrombin and PAR2 expression was increased in high proliferating cancer (p Overall (OS) and disease-free survival (DFS) PAR1 stromal expression was an independent predictor of reduced OS (HR 3.3, 95% CI 1.3-8.3, p=0.01) but did not correlate with DFS. There was no association between epithelial PAR1 expression or epithelial or stromal TF, thrombin or PAR2 expression and DFS or OS. CONCLUSION Stromal upregulation of the thrombin pathway occurs in in-situ cancer, implying cancer-stromal communication at the pre-invasive stage. Stromal thrombin pathway components may have a role in the transition of pre-invasive to invasive cancer. Stromal (but not epithelial) thrombin pathway upregulation is associated with aggressive invasive breast cancer phenotypes and reduced survival. The thrombin pathway may provide a novel therapeutic target, particularly in ER negative, HER2 positive breast cancer. Citation Format: Hudhaifah Shaker, Nigel J Bundred, Harith Albadry, Sarah L Nicholson, Susan Pritchard, Karin Jirstrom, Goran Landberg, Cliona C Kirwan. The thrombin clotting pathway is upregulated in the stroma of pre-invasive breast cancer and further upregulated in aggressive invasive breast cancer phenotypes [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-08-30.

Abstract P1-01-26: Procoagulant biomarkers may direct axillary nodal surgery

Background: Preoperative accurate staging of the axilla (through identifying invasive breast cancers with subclinical nodal metastases) will reduce the need for repeat axillary surgery. Tissue factor (TF) is overexpressed in cancer and is shed into the circulation leading to activation of the thrombin (extrinsic clotting) pathway. D-dimer is a fibrin degradation product and reliable systemic marker of thrombin pathway activation. TF and d-dimer are raised in breast cancer and may act as biomarkers to identify invasive cancer patients with undiagnosed lymph node (LN) metastases requiring axillary nodal clearance. Methods: Plasma d-dimer and TF were measured pre-operatively using ELISA in patients undergoing curative surgery and sentinel node biopsy (SNB) for invasive breast cancer (n = 125). D-dimer was considered to be raised if it was above the clinically used upper limit of normal of 500ng/ml and if TF was above 200pg/ml. D-dimer and TF were correlated with the presence or absence of axillary metastases at sentinel node biopsy. Results: Median age was 61 years (range 24–84). Mean invasive tumour size was 16.7 mm (range 1.6–70). One hundred and eleven (79%) invasive cancers were oestrogen receptor positive, 93 (67%) were progesterone receptor positive and 17(12%) were HER2 receptor positive. Pre-operative d-dimer was higher in LN positive (mean 599 ng/ml, 95% CI 415–864, n=32) versus LN negative (454ng/ml, 95% CI 400–514, n=93, p = 0.068) invasive cancer. TF was not significantly higher in LN positive cancer (vs LN negative), although TF did show weak correlation with d-dimer (r = 0.2, p = 0.03). Sixty eight patients (54%) had raised TF and 59 (47%) had raised d-dimer. On multivariate analysis (age, tumour size, invasive grade, ER status and presence of lymphovascular invasion as covariates) a raised pre-operative d-dimer combined with a raised pre-operative TF was a significant independent predictor of node positivity (odds ratio 6.43, CI 1.70–24.27, p = 0.006). Markers of systemic thrombin pathway activity (i.e. a combination of plasma d-dimer and TF) had a sensitivity of 91%, specificity of 32%, positive predictive value of 32% and negative predictive value of 92%. Conclusion: High levels of clotting activation are seen in breast cancer patients. Over 90% of patients with no axillary lymph node metastases had normal pre-operative levels of plasma TF and d-dimer. Combination of systemic markers of thrombin pathway activation may help predict axillary nodal involvement. The role of thrombin pathway activation in breast cancer metastasis is under ongoing investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-01-26.

P4-08-04: Preoperative Thrombin Pathway Activation Identifies Node Positive Patients in Early Breast Cancer.

Activation of the thrombin pathway (TP) is seen in 50% of cancers and plays a significant role in promoting metastasis. Cancer-induced TP activation is exacerbated by surgery, and this activation may be greater in the presence of metastases. Our aim was to determine if pre-operative plasma d-dimer, a marker of TP activation, correlated with the presence of LN metastases in early breast cancer. We also examined whether surgery-induced TP activation is affected by tumour-associated factors. Methods Plasma d-dimer was measured using automated ELISA pre-operatively and at days 1 and 42 following surgery in 103 prospectively recruited patients undergoing surgery for early breast cancer(87 invasive,13 DCIS). D-dimer values were log-transformed and correlated with LN metastases, tumour size and grade and hormone receptor status using parametric statistical tests. Results Median age was 68 years (range 46–82). Mean tumour size was 14.6 mm (range 7.5-40). Seventy invasive cancer patients had wide local excision(WLE) and sentinel node biopsy(SNB) and 17 had mastectomy(MX) +/− axillary clearance(ANC). Twenty-two patients had positive lymph nodes at primary surgery (MX 8,WLE 14). Of these 3(14%) were ER-ve and 19(86%) were ER+ve. Mean pre-operative d-dimer was higher (p Post-operative d-dimer was higher (p Conclusion Pre-operative TP activation and the thrombotic response to surgery (as measured by d-dimer) is greater in the presence of LN metastases regardless of tumour size. ER negative cancers (7/8 of which were LN negative) also resulted in a greater d-dimer rise, suggesting a difference in the thrombotic response to surgery between different cancer phenotypes. Ongoing work will determine if peri-operative TP activation can act as an independent predictor of node status and cancer recurrence. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-08-04.