Twenty-seven patients were recruited, eighteen in a non-randomised manner as part of a phase two, single arm, and single-centre study of gemcitabine plus intravenous ω-3FAs in patients with chemotherapy-naïve advanced pancreatic cancer. This trial was registered with cinicaltrials.gov: NCT01019382. Nine subsequent control patients who had received standard gemcitabine monotherapy were also recruited in a non-randomised manner. The local Ethics Committee and the Medicines and Healthcare Products Regulatory Agency (MHRA) approved both studies. All patients had a histological diagnosis of APC. All patients were discussed at the local multi-disciplinary team meeting and were assessed by an oncologist and considered suitable to receive gemcitabine chemotherapy. All patients were assessed against trial protocol inclusion/exclusion criteria.
Treatment protocol
Patients received a standard dose of gemcitabine (1000 mg/m2) administered as a thirty minute infusion once weekly for three weeks, followed by a one week break from treatment up to a maximum of six months. Immediately following each administration of gemcitabine patients received up to 500 mL of a lipid emulsion intravenously (Lipidem, BBraun) containing 10 g ω-3 fatty acids (0.5-1 g ALA and 4.3–8.6 g EPA/DHA) over four hours and all patients received at least 250 mL of Lipidem™. Control patients received gemcitabine treatment alone at a standard dose prescribed weekly by an oncologist with dose adjustments as per standard clinical practice. Patients were included in the study until progression of disease, death, serious adverse events necessitating withdrawal or patients withdrawing from treatment. A CT scan was performed at baseline, at any stage if disease progression was suspected, or every eight weeks. Patients continued in the trial as long as their tumour did not demonstrate progression as defined by the modified Response Evaluation Criteria in Solid Tumours (RECIST) criteria which was assessed by an independent radiologist. Blood samples were obtained at each treatment time point and kept on ice prior awaiting sample processing which occurred immediately following collection.
PMBC separation, storage and preparation
Blood samples consisting of 19.6 mL of whole blood were collected in 4 × 4.9 mL Ethylenediaminetetraacetic acid (EDTA) bottles from each patient at each treatment point following the completion of the treatment. Peripheral blood mononuclear cells (PBMCs) were isolated using standard protocols with a density gradient separation solution (Ficoll-Paque™ PREMIUM 1.084) and stored in an -80 °C freezer. Samples were snap-thawed at 37 °C in a temperature controlled water bath. The samples were washed and isolated and the cell number and percentage of viable cells was then determined prior to antibody staining using the cell counting protocol.
Sample staining and Flow cytometry analysis
PBMCs were analysed for EPCs. EPCs with three antibody phenotypes were analysed: 1. CD45−, CD31+ and CD133+, 2. CD45−, CD31+and CD34+ and 3. CD45−, CD31+, CD133+ and CD34+. Antibodies utilised to identify EPCs included CD31 FITC Mouse Anti-Human. BD Pharmingen®, CD45 Pacific Blue Mouse Anti-Human. BD Pharmingen® (Berkshire, UK), CD34 PE Mouse Anti-Human. BD Pharmingen® and CD133 APC. Miltenyi Biotec® (Surrey, UK). Samples were analysed for unstained cells, each individual antibody and a combination stain. Cells were processed using standard protocols. Samples were analysed with the FACS Aria II flow cytometer (Becton Dickinson, BD Biocsiences, San Jose, USA). Each sample was analysed for the unstained cells, each individual antibody evaluation and the overall combination analysis (Fig. 2).
Figure 2. FACS schematic scatter plots of EPCs with a quadruple stain for CD45−, CD31+, CD133+ and CD34+ antibodies. The top left plot shows side scatter (SSC) versus forward scatter (FSC). A mean of 374,796 cells in the target population (P1) were analysed over the one hundred and thirty-four samples investigated. Three populations of EPCs were analysed. EPCs with a CD45−, CD31+, CD133+ and CD34+ phenotype are seen in Q2.2 (CD133+ (APC) and CD34+ (PE)), gated off P6 (CD31+ (FITC) & CD45− (R/B220 pacific blue). EPCs with a CD45−, CD31+and CD34+ phenotype are seen in Q2.1 (CD34+ (PE) and CD31+ (FITC)), gated off P3 (CD45− (R/B220 pacific blue). EPCs with a CD45−, CD31+and CD133+ phenotype are seen in Q2 (CD31+ (FITC) and CD133+ (APC)), gated P3 (CD45− (R/B220 pacific blue).
Statistical analysis
Overall and progression free survival data was analysed with Kaplan-Meir curves and a log-rank (Mantel-Cox) test. Clinical outcomes were correlated with changes in mediators and survival curves analysed with a log-rank (Mantel-Cox) test. Changes in cells over the trial in both trial and control patients and between groups were analysed with logistic regression analysis using STATA software. A mixed effects logistic regression model was utilised that allowed for random variation and missed time points. These statistical models were chosen as the clinical trial data varied in length depending on individual treatment, in addition to missing time points resulting from the unavoidable factors, both patient and investigator related, in a clinical trial.
EPC measurement
EPCs are rare cells and make up between 0.01% and 0.001% of mononuclear cells in normal peripheral blood(19). The number of circulating CD34+ cells is around 50–100 per million white blood cells (0.005 − 0.001%), equal to about 350–700 cells per mL(20), and there are some report that suggest co-expression of CD133 increases the specificity for EPCs as it is not expressed on mature endothelial cells(20, 21). This co-expression of CD34 and CD133 cells in peripheral blood is even lower and makes quantification significantly more difficult. In order to compensate for this, most researcher groups acquire large amounts of cells or events. Strict criteria for the separation of cells, titrating of antibodies and the use of high-end flow cytometry machines and methods are essential. No universal agreement regarding phenotypic identification and lack of methodological consensus compounds the variability in the published literature. As a consequence there is wide variability in reported phenotypic subtypes that include a variety of markers (Table 1). There are also inconsistencies in the reporting of results, with some studies reporting EPCs for a sample volume and EPCs for a defined number of mononuclear cells(22). EPCs in this study were separated, processed, analysed and identified according to literature standards. EPCs are reported as a percentage of the parent population and three EPC phenotypes were examined.
Table 1
Antibody phenotypes and characteristics from select studies.
Study | Pathology | Subjects | Antibody |
| | | CD45- | CD31+ | CD133+ | CD34+ | CD146- | VEGFR2 |
Vizio et al (2010)8 | PAC | Human | Y | Y | Y | Y | | |
Sakamori et al (2012)54 | NSCLC | Human | Y | Y | Y | Y | | |
Morita et al (2011)55 | NSCLC | Human | Y | Y | Y | Y | | |
Steurer et al (2008)56 | NSCLC | Human | Y | Y | Y | Y | | |
Roodhard et al (2010)57 | Various* | Human | Y | Y | Y | | | |
Li et al (2011)58 | PAC | Mouse | | | Y | Y | Y | |
Staringer et al (2011)59 | PAC | Human | Y | Y | | | Y | |
Kuo et al (2012) | Breast | Human | Y | Y | Y | | Y | |
Lin et al (2013)60 | Rectal cancer | Human | Y | Y | Y | | | Y |
Fuereder et al (2014)61 | Prostate cancer | Human | Y | Y | Y | | Y | |
DuBois et al (2012)62 | Osteosarcoma | Human | Y | Y | Y | | Y | |
Corsini et al (2012)63 | Glioma | Human | Y | | Y | Y | | |
Kim et al (2013)64 | Gynaecological cancers | Human | Y | | Y | Y | | Y |
Bhatt et al (2011)65 | Renal cell carcinoma | Human | Y | | Y | Y | Y | |
Ha et al (2013)66 | Gastric cancer | Human | | | Y | Y | | |
Marlicz et al (2016)67 | Colorectal cancer | Human | Y | | Y | Y | | Y |
Starzynske et al (2013)40 | PAC | Human | Y | Y | | Y | | Y |
Ko et al (2010)42 | PAC | Human | Y | Y | | Y | | |
Steurer et al (2008)56 | NSCLC | Human | Y | Y | | Y | | Y |
Shim et al (2015)68 | Myocardial infarction | Human | Y | Y | | Y | Y | |
Mancuso et al (2011) | Breast cancer | Human | Y | Y | | Y | | |
Table 1. Demographic characteristics of the study participants and the number of treatment time points and completed cycles per group. A treatment cycle included three treatment points (each week) and a rest week.
Table 1. Antibody phenotypes and characteristics from select studies. There is wide discrepancy in the antibody phenotypes used to identify EPCs. In PAC the antibody phenotype of CD45−, CD31+, CD34+ & CD133+ 8 and CD45−, CD31+ and CD34+ 40,42. However the phenotype of CD45−, CD34+ & CD133+ has been investigated in various other cancers as shown above. NSCLC: Non-small cell lung cancer. VEGFR2: Vascular endothelial growth factor receptor 2. PAC: Pancreatic adenocarcinoma. Various*: Cancers studied include breast, colorectal, ovarian, oesophagus, prostate, head and neck, sarcoma, cervical and others not described. CD45: A hematopoietic marker also known as the Leukocyte Common Antigen which is present on all human leucocytes, including lymphocytes, monocytes, granulocytes, eosinophils and thymocytes. It is absent from erythrocytes, platelets or mature erythroid cells of bone marrow and non-haemopoietic tissues. CD31: An endothelial cell marker also known as PECAM-1 (Platelet And Endothelial Cell Adhesion Molecule 1), It is implicated in angiogenesis 69, vascular wound healing and transendothelial migration of leukocytes in inflammatory processes. It is widely expressed on endothelial cells as well as platelets, monocytes and granulocytes. CD34: An endothelial cell marker also known as the hematopoietic progenitor cell antigen, it is expressed on hematopoietic progenitor cells, vascular endothelium and some tissue fibroblasts. CD133: An early hematopoietic stem cell marker, CD133 is expressed on circulating endothelial progenitor cells21,31. In the hematopoietic system, CD133 expression is restricted to a subset of CD34 bright stem and progenitor cells in human foetal liver, bone marrow, cord blood and peripheral blood 70. CD146: Also known as melanoma cell adhesion molecule or MCAM it belongs to the immunoglobulin superfamily. It is expressed in epithelial cells, activated T cells, endothelial cells and multipotent mesenchymal stromal cells71. VEGFR-2: An endothelial marker.