Abstract
Aim: To investigate the relation between intima-media thickness (IMT) and laboratory parameters of atherosclerosis risk in patients with breast carcinoma. Patients and Methods: IMT and a panel of laboratory parameters associated with the risk of atherosclerosis were studied in 192 patients with histologically-verified breast carcinoma. Results: Patients with metastatic disease had significantly higher fibrinogen, C-reactive protein (CRP), urinary neopterin and mean IMT, and significantly lower serum albumin and hemoglobin concentrations. Significant correlations were observed between CRP, urinary neopterin, mean IMT and other parameters of cardiovascular risk. Age was an independent predictor of the presence of sonographic signs of atherosclerosis using logistic regression, and age, glucose, time from start of chemotherapy, high-density lipoprotein cholesterol, D-dimers were independently associated with IMT in stepwise regression models. Conclusion: In addition to the associations between IMT and laboratory or clinical parameters of the risk of atherosclerosis, IMT may also be associated with the time from chemotherapy.
Recent decades have witnessed major progress in cancer therapy, which can be translated into improved survival of patients with different types of primary tumors. With this improved survival, and even cure of patients with some forms of metastatic cancer, hitherto unknown and unexpected long-term complications have emerged, including atherosclerosis and associated disorders (1-3). Comorbid conditions are now an important issue in medical oncology, and, in many patients, comorbid disorders rather than cancer remain the ultimate cause of death (4). Atherosclerosis, with its complications, is by far the most important cause of morbidity and mortality due to comorbid conditions in patients with cancer. Age, smoking, obesity and oxidative stress are associated with increased risk of atherosclerosis, as well as many forms of cancer (5). Several retrospective series demonstrated increased risk of cardiovascular events, e.g. myocardial infarction, in long-term survivors of testicular cancer or of pediatric tumors treated with chemotherapy (1-3). The toxicity of anticancer therapy may result in the progression of atherosclerosis (6). The information on the prevalence of complications of atherosclerosis is limited for most of the common types of cancer (5, 7-9). In patients with breast carcinoma both increased and decreased incidence of complications of atherosclerosis have been reported (10, 11).
More pronounced adverse effects fostering the progression of atherosclerosis could be expected from the use of targeted agents, e.g. drugs targeting the vascular endothelial growth factor or its receptors, but these drugs were introduced only recently and are now used almost exclusively for patients with incurable metastatic tumors. For some targeted agents, hypertension, hyperlipidemia, or hypomagnesemia represent the most important side-effects. Hypertension or proteinuria accompanying administration of drugs targeting vascular endothelial growth factor is associated with endothelial dysfunction (12). With the introduction of targeted agents into the standard therapy of common tumor types, e.g. breast carcinoma or colorectal carcinoma, the effect of treatment on long-term risk of atherosclerosis may become an important consideration.
Considerable efforts in the past several decades have been devoted to identifying risk factors for the development or early signs of the presence of atherosclerosis. A number of laboratory parameters, including cholesterol, homocysteine, and C-reactive protein (CRP) (13), have been shown to predict cardiovascular events associated with atherosclerosis. In addition to the search for laboratory parameters of increased risk, strategies have been developed for early diagnosis of asymptomatic cardiovascular disease. Identification of risk or early diagnosis of asymptomatic disease opens the possibility of intervention that could prevent a cardiovascular event. Stress/rest myocardial perfusion scan, and measurement of carotid intima-media thickness (IMT) have been the most widely used diagnostic tests in asymptomatic individuals. Stress/rest myocardial perfusion has been shown to predict cardiovascular events in asymptomatic patients with different risks of coronary artery disease (14, 15). Measurement of carotid IMT has been established as an indirect non-invasive test for the detection of coronary atherosclerosis (16).
The aim of the present study was to investigate the relation between IMT and laboratory parameters of atherosclerosis risk in patients with breast carcinoma.
Patients and Methods
One-hundred and ninety-two female patients with histologically-verified breast carcinoma, aged (mean±standard deviation) 54±10 (range 28-76) years, were included in the present study. The investigations were approved by Institutional Ethical Committee and the patients signed an informed consent.
Body-mass index (BMI) was calculated using the formula: weight (in kg)/[height (in m)]2. The menopausal status; history of breast cancer, including the time from diagnosis, time from chemotherapy start or the presence of distant metastases; history of smoking, cardiac disorder, hypertension, diabetes, disorders of lipid metabolism and thyroid disorders were recorded. Blood samples were collected from a peripheral vein after an overnight fast. The samples were transferred immediately to the laboratory, centrifuged (1600 ×g, 10 min at 16°C), then the serum was separated and either processed immediately (for most parameters) or frozen at −20°C until analysis (for alpha-tocopherol and retinol). Peripheral blood cell count was performed as described elsewhere (17). Hemoglobin was measured by a photometric method using sodium lauryl sulfate; leukocytes and platelets were determined by an impedance method using a Sysmex XE-2100 blood analyzer (Sysmex, Kobe, Japan). Fibrinogen was determined by Clauss clotting time method, using commercially available reagents (Grifols, Barcelona, Spain). Antithrombin functional activity was measured by the spectrophotometric method using chromogenic substrates (STA Compact; Diagnostica Stago, Paris, France), and D-dimers in plasma were determined by an immunoturbidimetric method (Diagnostica Stago).
CRP was determined using particle-enhanced immunoturbidimetric assay; lipoprotein (a), serum cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides were determined using immunoturbidimetric and enzyme-based assays accordingly, as commercially available on a MODULAR analyzer (Hoffmann-La Roche, Basel, Switzerland). Homocysteine concentration was determined immunochemically (Immulite 2000; Siemens Healthcare Diagnostics, Deerfield, IL, USA). Glycosylated hemoglobin was determined by high-performance liquid chromatography using a Variant II Turbo System (Bio-Rad, Hercules, CA, USA) according to the instructions of the manufacturer and expressed according the International Federation of Clinical Chemistry (IFCC) reference system (18). Serum glucose, creatinine, magnesium, uric acid and albumin were determined using commercially available kits on a MODULAR analyzer (Hoffmann-La Roche).
Serum alpha-tocopherol and retinol were determined by high-performance liquid chromatography, as described elsewhere (19). In a liquid-liquid extraction procedure, 500 μl of serum were de-proteinized by cool ethanol denatured with 5% methanol (500 μl, 5 min at 4°C). Subsequently, 2.5 ml of n-hexane were added to this mixture and extraction for 5 min was carried-out by a vortex apparatus. After centrifugation (1 600 ×g, 10 min at 0°C), the aliquot (2 ml) of the extract was separated and reduced to dryness (45°C) in a vacuum concentrator (AD 5301; Eppendorf, Hamburg, Germany). The residue was dissolved in 400 μl of methanol and analyzed by reversed-phase high-performance liquid chromatography using external standard calibration. The analyses were performed using a Perkin-Elmer (Norwalk, CT, USA) high-performance liquid chromatography set comprising of an LC 200 pump, an LC 200 autosampler, LC column oven 101 thermostat and LC 235C diode array detector, attached to a Perkin-Elmer Turbochrom Chromatography Workstation version 4.1. Separation of alpha-tocopherol and retinol was performed using Chromolith Performance RP-18e, 100×4.6 mm monolithic columns (Merck, Darmstadt, Germany). As the mobile phase, 100 % methanol was used at a flow rate of 2.5 ml/min and column pressure of 3.3 MPa. The block heater LC Oven 101 (Perkin-Elmer) was utilized to maintain the analytical column temperature at 25°C. The sample injection volume was 50 μl. The detection of alpha-tocopherol and retinol was carried out at 295 nm and at 325 nm, respectively. Limit of detection (LOD) and limit of quantification (LOQ) of alpha-tocopherol were 0.1 μmol/l and 0.3 μmol/l, respectively. LOD and LOQ of retinol were 0.02 μmol/l and 0.07 μmol/l, respectively.
Early-morning urine samples were collected and stored at −20°C until analysis. Urinary neopterin was determined using a modification of a method described earlier (20). After centrifugation (45 s, 12000 ×g), 100 μl of urine specimen were diluted with 1.0 ml of mobile phase containing 2 g of disodium-EDTA per liter; the samples were then filtered using a Microtiter, AcroPrep 96 Filter Plate 0.2 μm/350 μl vacuum manifold (Pall Life Science, Ann Arbor, MI, USA). Neopterin was determined using a Prominence LC20 high-performance liquid chromatography system (Shimadzu, Kyoto, Japan) composed of Rack changer/C autosampler for micro-titration plates, DGU-20A5 degasser, 2 LC-20 AB liquid chromatograph pumps, auto sampler SIL-20 AC, column oven CTO – 20 AC thermostat, RF-10 AXL fluorescence detector, SPD – M20A diode array detector and CBM-20A communications bus module. Phosphate buffer 15 mmol/l, pH 6.4, with flow rate of 0.8 ml/min was used as the mobile phase. Separation was performed using a Gemini Twin 5 μ, C18, 150×3 mm (Phenomenex, Torrance, USA) hybrid analytical column at 25°C, with an injection volume of 1 μl. Neopterin was identified by its native fluorescence (353 nm excitation, 438 nm emission wavelength). Creatinine was monitored simultaneously in the same urine specimen with diode array detector at 235 nm. Time of analysis for urine neopterin and creatinine was 6 min and the analytes were quantified by external standard calibration. LOD and LOQ of urinary neopterin were 5.89 nmol/l and 11.79 nmol/l, respectively. LOD and LOQ of urinary creatinine were 6 μmol/l and 12 μmol/l, respectively. Neopterin concentrations are expressed as neopterin/creatinine ratio (μmol/mol creatinine).
The urinary albumin concentration was measured immunochemically using COBAS-MIRA (Hoffmann-La Roche), and expressed as the albumin/creatinine ratio (g/mol creatinine). LOQ of urinary albumin was 3.6 mg/l. The activity of N-acetyl-β-D-glucosaminidase (NAG) in urine was determined using a method previously published (21), with minor modifications. LOQ of urinary NAG was 1 nkat/l. NAG activity is expressed as NAG/creatinine ratio (μkat/mol creatinine).
The carotid arteries were evaluated with two-dimensional imaging using an ultrasound scanner (Aplio, Toshiba, Tokyo, Japan) with an 8-14 MHz transducer (22). IMT, defined as the distance between the echogenic line representing blood–intima interface and the echogenic line representing media-adventitia interface, was measured on the posterior wall of the carotid artery in the longitudinal plane. IMT was evaluated bilaterally at two levels, in the supraclavicular segment of the common carotid artery and 2 cm below the bifurcation of the common carotid artery (23). Three measurements were performed at each level and the mean of these three measurements was calculated. The presence of the carotid plaque was noted. The measurements were performed by the same observer. The mean IMT and maximal IMT from the measurements performed bilaterally at the two segments were determined. Sonographic signs of atherosclerosis were considered present if the IMT was ≥1 mm or if presence of plaques or calcifications was observed.
Differences between groups of patients were analyzed by the Mann-Whitney U-test. Correlations were analyzed using the Spearman's rank correlation co-efficient. These analyses were performed using the NCSS software (Number Cruncher Statistical Systems, Kaysville, UT, USA). Multivariate analysis was performed using multiple logistic regression and multiple regression. These analyses were performed using the SAS software (Version 9.2; SAS Institute, Cary, NC, USA). The decision on statistical significance was based on p=0.05 level.
Results
Fifteen patients had metastatic disease and 177 patients had no evidence of distant metastases. Patients with metastatic disease had significantly higher fibrinogen, CRP, urinary neopterin and mean IMT, and significantly lower serum albumin and hemoglobin (Table I).
Correlations between CRP, urinary neopterin, mean IMT and other parameters of cardiovascular risk are shown in Table II. Significant positive correlations were observed between IMT and age, BMI, D-dimers, fibrinogen, glucose, uric acid, total cholesterol, LDL cholesterol, triglycerides, homocysteine, glycosylated hemoglobin, retinol, CRP, urinary NAG and albumin, and negative correlations were observed between IMT and HDL cholesterol and albumin (Table II). Significant negative correlations were observed between urinary neopterin and hemoglobin, platelet count, creatinine, HDL cholesterol, albumin, retinol and alpha-tocopherol. Significant positive correlations were observed between CRP and age, BMI, leukocyte count, D-dimers, fibrinogen, glucose, uric acid, triglycerides, urinary albumin and both mean and maximal IMT. CRP correlated negatively with HDL cholesterol and albumin.
Thirty-five patients (18%) had sonographic signs of atherosclerosis, as defined in the Patients and Methods, while no signs of atherosclerosis were evident in 157 patients. The patients with sonographic signs of atherosclerosis had significantly higher age, BMI, fibrinogen, glucose, total cholesterol, LDL cholesterol, triglycerides and glycosylated hemoglobin, and significantly lower HDL cholesterol (Table III).
The continuous clinical and laboratory parameters were then dichotomized based on the upper limit of normal values (D-dimers, fibrinogen, glucose, uric acid, total cholesterol, LDL cholesterol, lipoprotein (a) and CRP); the lower limit of normal values (serum albumin), or, in the case of parameters with fewer than 10% of values above (or below) the normal range or for those for which the normal range was difficult to define, above the third quartile (age, BMI, leukocyte and platelet count, antithrombin, creatinine, triglycerides, homocysteine, glycosylated hemoglobin, retinol and alpha-tocopherol, urinary NAG, albumin and neopterin) or below the first quartile (hemoglobin, magnesium and HDL cholesterol).
A multiple logistic regression using a stepwise selection model was subsequently performed for data from 149 patients who had complete dichotomized data for all variables. Only age was an independent predictor of the presence of sonographic signs of atherosclerosis (p<0.0001), and the probability (P) of atherosclerosis was defined by the formula: P=1/[1+exp (−12.8297+0.1952×age)]. Using continuous variables, multiple regression analyses were then performed using log mean IMT or log maximum IMT as dependent variables. Using a stepwise selection model, increasing age (p<0.0001), glucose (p=0.0238) and time from chemotherapy start (TFCS; p=0.0205) were independently associated with increased mean IMT, while higher HDL was associated with decreased IMT (p=0.0458). The mean IMT was defined by the formula: ln (mean IMT)=−1.35740+0.01469×age+ 0.02933×glucose+0.001054× (TFCS–0.05715)×HDL.
Using the same stepwise selection procedure, increasing age (p<0.0001), and glucose (p=0.0324) were independently associated with increased maximum IMT, while D-dimers (DD; p=0.0297) and HDL (p=0.0291) were independently associated with decreased maximum IMT. Maximum IMT was defined by the formula: ln (maximum IMT)= −1.28323+0.01611×age–0.01871×DD+0.03261×glucose−0.07342×HDL.
The investigated parameters in patients with and those without distant metastases.
Discussion
In a breast cancer patient population IMT, an indicator of the presence of atherosclerosis, was associated with risk factors of atherosclerosis that are well-known from the general population, including age, BMI, glucose, uric acid, HDL cholesterol, LDL cholesterol, albumin and homocysteine (24). An association was also evident with the systemic inflammatory response. CRP, a key indicator of systemic inflammatory response, was correlated with IMT, as well as with risk factors of atherosclerosis, including BMI, uric acid or HDL cholesterol. In multivariate analysis using logistic regression, only age predicted the probability of atherosclerosis. When applying the stepwise multiple regression model, age, glucose, HDL, time from the chemotherapy start and D-dimers were independent predictors of mean or maximum IMT, while age, glucose, and time from the start of chemotherapy were associated with increased IMT; HDL and D-dimers had a protective effect. Interestingly, a positive correlation was also observed between IMT and D-dimers, indicating that the role of fibrinolysis may be more complex in this patient population. The association between IMT and the time from chemotherapy start is of specific interest. In many patients this time was zero as they were not treated with chemotherapy. The association with IMT indicating that the risk of atherosclerosis increases with the time elapsed from the chemotherapy start corresponds to the hypothesis that cytotoxic chemotherapy plays an important role in the pathogenesis of atherosclerosis in patients with cancer (1-3, 6).
Interestingly, IMT was increased in patients with distant metastases. Other parameters that were different in patients with metastatic disease included parameters associated with systemic inflammatory response, urinary neopterin, CRP, fibrinogen and hemoglobin. The observed association of increased IMT with distant metastasis and inflammatory response opens up the possibility that the systemic inflammatory response that is linked with advanced disease may induce accelerated atherosclerosis. This association would not be surprising if we consider data linking inflammatory disorders and atherosclerosis (25-27). For example, in an earlier study, progression of IMT was associated with infectious burden (28). Moreover, it is now generally accepted that atherosclerosis is an inflammatory disorder (29). Parameters of the systemic inflammatory response, e.g. serum CRP, are predictive of the risk of cardiovascular events (13, 30-32). CRP is increased in both patients with advanced cancer, as well as in patients with atherosclerosis, and advanced cancer is often a more potent factor inducing a systemic inflammatory response than are benign infections or inflammatory disorders. Neopterin, a product of activated macrophages, is also increased in patients with cancer, and cardiovascular disorders (33, 34), and increased neopterin concentrations were shown to be associated with cardiovascular and all-cause mortality (35, 36). Although the association of increased IMT and distant metastasis was not confirmed in multivariate analysis, only a small proportion of patients in the present cohort had distant metastasis, and the association between metastasis and IMT should be studied in a larger cohort.
Correlations between C-reactive protein (CRP), urinary neopterin, intima-media thickness (IMT) and other parameters of atherosclerosis risk.
The data on association between cancer and atherosclerosis that would be independent of the effect of anticancer therapy are very limited (5, 7-9). This is understandable since the risk factors of atherosclerosis may particularly be increased in patients with advanced or metastatic tumors that are almost invariably lethal in the absence of systemic anticancer therapy. In fact, only few metastatic tumors are curable with intensive chemotherapy, and for these patients it is difficult or even impossible to separate the effects of therapy from the effects of systemic phenomena associated with advanced cancer. The incidence of most tumor types increases with age. The incidence of atherosclerosis is also age-related, presenting another confounding effect when analyzing the association between cancer and atherosclerosis. In a 1978 review on coronary artery disease in patients with cancer, Kopelson and Herwig (7) stated that coronary artery disease is rare in such patients. Obviously, this review covered an era when no metastatic cancers were curable. Most of the data on increased incidence of cardiovascular events associated with atherosclerosis come from studies performed on tumors that affect children and young adults,who live long after the cure to allow for the manifestation of these chronic complications (1-3). IMT is markedly increased after neck irradiation (37). Increased risk of stroke has been observed in patients with early breast cancer treated with radiotherapy (11). However, the use of tamoxifen has been associated with a reduction of IMT (38). In addition to hypertension or hypercholesterolemia, anticancer therapy or advanced cancer itself may affect some more recently discovered laboratory parameters, associated with the progression of atherosclerosis, including the systemic inflammatory response and antioxidant balance.
The investigated laboratory and clinical parameters in patients with and those without sonographic evidence of carotid atherosclerosis.
In patients with cancer and evidence of atherosclerosis, it is difficult to discern the causal sequence of events, i.e. whether the increase of inflammatory parameters merely reflects associated atherosclerosis, or whether the inflammatory response induced by cancer leads to accelerated atherosclerosis. Present data indicating the presence of multiple correlations of parameters of lipid metabolism and other clinical and laboratory parameters associated with the risk of atherosclerosis in patients with breast carcinoma may be difficult to interpret with regard to the causal relationship in the pathogenesis of atherosclerosis. Similarly to a general population (39), an inverse correlation was observed between neopterin and antioxidant vitamins in patients with breast cancer.
In conclusion, the associations between the presence of sonographic signs of atherosclerosis or IMT and laboratory or clinical parameters are similar to those of the general population, but the association with chemotherapy suggests that treatment could also be a significant risk factor in this population. The association with the presence of distant metastases should be further explored.
Acknowledgements
Supported by the grants of the Internal Grant Agency of the Czech Republic NT/13564 and NR9096-4, and the project Biomedreg CZ.1.05/2.1.00/01.0030.
- Received May 21, 2012.
- Revision received July 24, 2012.
- Accepted July 25, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
