Levels of Circulating Alternatively Spliced Tissue Factor (asTF) in the Plasma of Patients with Pancreatic Ductal Adenocarcinoma (PDAC) May Help Predict Aggressive Tumor Phenotype

Tissue Factor (TF) present in blood cells and plasma is referred to as blood-borne or circulating TF. TF has been implicated in the pathogenesis of several chronic disease states, most notably cardiovascular disease/thrombosis, diabetes, and cancer. Full-length TF is an integral membrane protein while alternatively spliced TF can be secreted in a free form and features a unique C-terminal domain enabling its selective detection in bio-specimens. Recently, asTF was shown to circulate in the blood of metastatic breast cancer patients at concentrations exceeding 1 ng/mL (Kocaturk et al, PNAS 2013), and it promoted tumor growth and spread in an orthotopic model of pancreatic ductal adenocarcinoma (PDAC, Unruh et al, Int J Cancer, 2014). asTF protein acts as a cell agonist driving angiogenesis, cancer cell proliferation, and monocyte recruitment via integrin binding. It is not known whether circulating asTF may contribute to or serve as a biomarker in patients suffering from cardiovascular disease, diabetes, and/or solid cancers including PDAC. We evaluated circulating asTF in healthy subjects and individuals with ongoing acute coronary syndrome (ACS); diabetes mellitus (DM); ongoing ACS+DM; and PDAC.

Samples of platelet poor plasma from 204 subjects were obtained from University of Cincinnati Cancer Institute’s Tumor Bank and Diagnostica Stago collections, blood specimens drawn from emergency room visitors at four medical centers in the US, and George King Bio-Medical, Inc. ACS was defined by positive troponin levels; DM was self-identified. Blood was drawn into tubes containing heparin (ASC, DM, ACS+DM), acid citrate dextrose (PDAC), or sodium citrate (healthy subjects), centrifuged at 3000 rpm for 15 min at 4°C, and stored at -80°C until use. Blinded asTF ELISA was performed on plasma samples as per the prototype-tailored procedure (Diagnostica Stago). Samples with asTF concentrations ≥0.2 ng/mL were deemed positive. asTF concentrations are presented as mean±SD. Kruskal-Wallis one-way analysis of variance was used to compare differences in concentration levels between the cohorts; Chi-Square and/or Fisher’s exact test were used to compare proportions.

asTF protein was detectable in the plasma of 3/19 (15.8%) subjects in the healthy cohort (CORE Set 50, George King Bio-Medical); 7/38 (18.4%) in the no ACS/no DM cohort (emergency room visitors’ control group); 2/40 (5%) in the DM cohort; 5/39 (12.8%) in the ACS cohort; 4/25 (16.0%) in the ACS/DM cohort; and 20/43 (46.5%) in the PDAC cohort; the proportion of PDAC patients positive for asTF was significantly higher compared to that in all other cohorts (p<0.01, Chi-Square test). The mean asTF concentrations in the cohorts were as follows: PDAC, 0.403±0.912 ng/mL; healthy subjects, 0.169±0.596 ng/mL; emergency room visitors’ control group, 0.159±0.357 ng/mL; ACS, 0.0925±0.258 ng/mL; DM, 0.0423±0.19 ng/mL; ACS+DM, 0.208±0.642 ng/mL; the differences between mean asTF levels in the cohorts did not reach significance. Next, we evaluated asTF’s potential as a biomarker to help detect a more aggressive PDAC phenotype. Among the 43 patients with PDAC, 36 were initially deemed resectable and 7 unresectable due to the presence of metastatic disease as determined by diagnostic screening; following exploratory laparoscopic surgery, 11 out of 36 patients initially deemed resectable were deemed unresectable due to the presence of metastatic disease. When the entire PDAC cohort was split into bona fide resectable (25) and unresectable (18) sub-cohorts, positivity for asTF was significantly more prevalent in the unresectable sub-cohort irrespective of the results of initial evaluation and/or pre-operative CA19-9 levels (asTF ≥0.2 ng/mL: 13 unresectable and 7 resectable patients; asTF<0.2 ng/mL: 5 unresectable and 18 resectable patients, p=0.0059, Fisher’s exact test).

We here report that asTF at levels ≥0.2 ng/mL occurs more frequently in the plasma of patients with PDAC compared to healthy subjects and/or individuals with ACS, DM, and ACS/DM. Further, PDAC patients whose plasma asTF levels were equal to or exceeded 0.2 ng/mL had a significantly lower chance to qualify for tumor resection, irrespective of initial pre-surgical diagnostic evaluation. asTF may thus comprise a novel marker of aggressive PDAC phenotype with potential utility in patient stratification, warranting prospective evaluation of larger PDAC patient cohorts.