Paul S. Stonelake

Plasma angiopoietin-1, angiopoietin-2 and Tie-2 in breast and prostate cancer: a comparison with VEGF and Flt-1.

Background  Angiogenesis is essential for tumour growth and metastasis, and is coordinated by several classes of growth factors mediating their effect through receptors linked, in turn, to tyrosine kinase. These growth factors include angiopoietin‐1 (Ang‐1), angiopoietin‐2 (Ang‐2) and vascular endothelial growth factor (VEGF), which act through receptors Flt‐1 and Tie‐2.

Detection and quantification of mature circulating endothelial cells using flow cytometry and immunomagnetic beads: A methodological comparison

Mature circulating endothelial cells (CECs) are novel cellular markers of endothelial damage/dysfunction. The two main techniques of CEC enumeration are flow cytometry (FC) and immunomagnetic bead (IB) isolation. Both quantify CECs accurately, but a direct comparison of both methods has not been reported. We sought to assess the agreement between the two methods in two patient populations, and a group of healthy subjects, with emphasis given to methodological issues. We included 34 patients with acute coronary syndrome (ACS), 60 patients with primary breast cancer (PBC) and 30 healthy controls (HC). We quantified CECs using the IB method [CD146 and FITCUlex europaeus lectin-1] and FC [CD45, CD34 and CD146]. Bland-Altman plots suggested reasonable agreement (<5% of events >2 standard deviations from the mean) between FC and the IB methods for CEC quantification in whole blood in the two disease groups (ACS and PBC), but not among the HCs. There were no statistically significant differences in CEC levels by the two methods amongst all three patient groups. There is reasonable agreement between the FC and the IB methods for mature CEC quantification in whole blood, especially amongst disease groups. The agreement between the two methods appears to weaken in healthy controls, and at lower and higher absolute CEC counts.

SELDI-TOF-MS determination of hepcidin in clinical samples using stable isotope labelled hepcidin as an internal standard

BackgroundHepcidin is a 25-residue peptide hormone crucial to iron homeostasis. It is essential to measure the concentration of hepcidin in cells, tissues and body fluids to understand its mechanisms and roles in physiology and pathophysiology. With a mass of 2791 Da hepcidin is readily detectable by mass spectrometry and LC-ESI, MALDI and SELDI have been used to estimate systemic hepcidin concentrations by analysing serum or urine. However, peak heights in mass spectra may not always reflect concentrations in samples due to competition during binding steps and variations in ionisation efficiency. Thus the purpose of this study was to develop a robust assay for measuring hepcidin using a stable isotope labelled hepcidin spiking approach in conjunction with SELDI-TOF-MS.ResultsWe synthesised and re-folded hepcidin labelled with 13C/15N phenylalanine at position 9 to generate an internal standard for mass spectrometry experiments. This labelled hepcidin is 10 Daltons heavier than the endogenous peptides and does not overlap with the isotopic envelope of the endogenous hepcidin or other common peaks in human serum or urine mass spectra and can be distinguished in low resolution mass spectrometers. We report the validation of adding labelled hepcidin into serum followed by SELDI analysis to generate an improved assay for hepcidin.ConclusionWe demonstrate that without utilising a spiking approach the hepcidin peak height in SELDI spectra gives a good indication of hepcidin concentration. However, a stable isotope labelled hepcidin spiking approach provides a more robust assay, measures the absolute concentration of hepcidin and should facilitate inter-laboratory hepcidin comparisons.

Use of multiple drains after mastectomy is associated with more patient discomfort and longer postoperative stay.

BACKGROUND Seromas constitute a common complication following surgery for breast cancer, and closed drainage is used routinely to reduce its incidence. The aim of this study was to evaluate the influence of number of drains on patient discomfort, seroma formation, and hospital stay during the immediate postoperative period after mastectomy for breast cancer. PATIENTS AND METHODS Based on a retrospective review of our clinical database, 110 consecutive patients from January 2004 through January 2006 who had undergone a mastectomy and axillary clearance for breast cancer were sent a simple postal questionnaire for collection of data. RESULTS A total of 70 patients responded (all women; mean age, 69.4 +/- 11.4 years). Twenty-seven patients (38.57%) had 3 drains implanted unilaterally, 24 (34.28%) had 2, and 19 (27.14%) had 1 drain. They were divided into 2 groups: the first group with 1 drain (19 patients) and the other with 2 or 3 drains (51 patients). Median postoperative hospital stay was 2 days (range, 1-8 days); patients with 1 drain had a significantly shorter postoperative hospital stay (median, 2 days [range, 1-4 days] vs. 2 days [range, 1-8 days]; Mann-Whitney U test, P = .02). A total of 15 patients (21.43%) complained of a seroma. There was no difference in seroma rates between groups. Patients who had a single drain implanted had a significantly lower rate of discomfort (median, 2 [range, 1-5] vs. 3 [range, 1-7]; Mann-Whitney U test; P = .04). CONCLUSION The number of drains used after a mastectomy for breast cancer did not significantly affect the rate or amount of seromas in this study, but the use of a single drain after mastectomy was significantly associated with less discomfort and shorter postoperative hospital stay.

Hypertension, Anti-Hypertensive Therapy and Neoplasia

The link between cancer, hypertension and anti-hypertensive drug treatment is controversial. Despite numerous studies looking either directly or indirectly at cancer and hypertension, the results are often conflicting and do little to answer the dominant questions of cause and effect. Also, the treatment of hypertension has continued to evolve, with newer therapies being made available including angiotensin-converting enzyme inhibitors. Whilst the potential link with cancer is thought to be small at worst, with the overall benefits of hypertension far outweighing its negative impacts, the suggestion of a carcinogenic role for either hypertension or its treatment continues to be an emotive issue, and needs firm answers. In this review, we provide an overview establishing the strengths and weaknesses of the arguments presented and highlight possible pharmacophysiological pathways involved.

Circulating endothelial cells in malignant disease

Cancer is a disease largely dependent on neoangiogenesis. Cancer neoangiogenesis is often disordered and abnormal, with evidence of coexisting vascular endothelial dysfunction. A novel method of assessing vascular endothelial function in cancer is via the quantification of circulating endothelial cells (CEC). Unusual in healthy individuals, their presence in elevated numbers often indicates substantial vascular endothelial perturbation. Another interesting cell type is the endothelial progenitor cell (EPC), whose numbers increase in the presence of vascular damage. Recent research suggests that EPCs have an important role in tumor vasculogenesis. Another marker being investigated in the context of vascular dysfunction and coagulopathy is the endothelial microparticle (EMP). Thus, CECs, EPCs and EMPs may represent potentially novel methods for evaluating the vascular status of cancer patients. This review will summarize the current position of CECs, EPCs and EMPs in cell biology terms, with particular emphasis on their relationship to malignant disease.

The angiome: a new concept in cancer biology

Angiogenesis is a key process in cancer biology, and justifies focus on the endothelium. Separate studies have looked at different aspects of angiogenesis and vascular biology, primarily focusing on certain laboratory and imaging techniques that generally reflect one particular aspect of the assessment of the endothelium. These techniques include the secretion/release of molecules (such as growth factors) into the plasma, by the presence of mature and progenitor endothelial cells themselves in the circulation, but also by examination of peripheral blood flow and the local circulation of the tumour, and cells of the tumour itself. However, a limitation of this approach is that these methods, although themselves being useful, have often been viewed in isolation and thus can provide only a part of the vascular picture. The authors submit that this approach is weak, and introduce ‘the angiome’ as a term which fuses several different aspects of endothelial and tumour biology into a single concept. The authors suggest that the adoption of the concept of the angiome will bring improved insights into angiogenesis and thus cancer cell biology. In justifying this concept, the authors review the current understanding of endothelial biology and the methods of its assessment, and hypothesise that a more multifactorial approach to the angiome will be a crucial determinant of outcomes of and treatment strategies for diseases, in particular antiangiogenics for cancer therapy.

A comparison of plasma versus histologic indices of angiogenic markers in breast cancer.

BackgroundOver-expression of angiogenic growth factors and their receptors, and high levels of these molecules in the blood, are a common feature of cancer although the relationships between cell expression and plasma levels are unknown. We hypothesized a significant correlation between the expression and cellular distribution of vascular endothelial growth factor (VEGF), its receptor Flt-1, and the angiopoietin receptor Tie-2 with levels of these molecules in the plasma. MethodsThe tissue expression of VEGF, Flt-1, and Tie-2 were investigated by immunohistochemistry, and plasma levels assessed by enzyme-linked immunosorbent assay in 36 patients with breast cancer and 15 with benign breast disease. ResultsDespite expected significant differences in plasma levels of the molecules (P<0.03 to <0.001), no significant differences were found in Tie-2, VEGF, and Flt-1 tissue expression between breast cancer and benign disease controls. No significant correlations were observed between plasma levels of their tissue expression. ConclusionsTissue expression of Tie-2, VEGF, and Flt-1 may not be an overly sensitive tool for assessing abnormalities of coagulation, platelet activation, and angiogenesis in human cancer. Plasma markers may not be representative of tumor activity, and may not come wholly from tumor cells. Instead these markers may be indicative of endothelial dysfunction in cancer patients.

Effect of standard chemotherapy and antiangiogenic therapy on plasma markers and endothelial cells in colorectal cancer

Introduction:The importance of the endothelium in angiogenesis and cancer is undisputed, and its integrity may be assessed by laboratory markers such as circulating endothelial cells (CECs), endothelial progenitor cells (EPCs), plasma von Willebrand factor (vWf), soluble E selectin, vascular endothelial growth factor (VEGF) and angiogenin. Antiantigenic therapy may be added to standard cytotoxic chemotherapy as a new treatment modality. We hypothesised that additional antiangiogenic therapy acts in a contrasting manner to that of standard chemotherapy on the laboratory markers.Methods:We recruited 68 patients with CRC, of whom 16 were treated with surgery alone, 32 were treated with surgery followed by standard chemotherapy (5-flurouracil), and 20 were treated with surgery followed by standard chemotherapy plus anti-VEGF therapy (Avastin). Peripheral blood was taken before surgery, and again 3 months and 6 months later. CD34+/CD45−/CD146+ CECs and CD34+/CD45−/CD309[KDR]+ EPCs were measured by flow cytometry, plasma markers by ELISA.Results:In each of the three groups, CECs and EPCs fell at 3 months but were back at pre-surgery levels at 6 months (P<0.05). VEGF was lower in both 3-and 6-month samples in the surgery-only and surgery plus standard chemotherapy groups (P<0.05), but in those on surgery followed by standard chemotherapy plus anti-VEGF therapy, low levels at 3 months (P<0.01) increased to pre-surgery levels at 6 months. In those having surgery and standard chemotherapy, soluble E selectin was lower, whereas angiogenin was higher at 6 months than at baseline (both P<0.05).Conclusions:We found disturbances in endotheliod cells regardless of treatment, whereas VEGF returned to levels before surgery in those on antiangiogenic therapy. These observations may have clinical and pathophysiological implications.

Endothelial progenitor cells: from pathophysiology to clinical practice

ume or the presence or absence of urodynamically proven bladder outlet obstruction was unknown, mirroring clinical practice, especially in a primary care setting. It is unique in that it reports the subjective improvement in heath-related quality of life achieved with OXY-TDS as measured by the KHQ, demonstrating both statistically and clinically significant changes. It is the first study to demonstrate improvements in depression and work productivity achieved with antimuscarinics in a large cohort of male patients. The literature and clinical experience substantiate the efficacy, tolerability and safety of OXY-TDS in the treatment of men and women with OAB. The unique patch delivery system and its low incidence of dry mouth and constipation position it as an excellent first-line therapy. There is no question that it is an important addition to the therapeutic armamentarium in the management of patients with OAB.