Does Chronic Low-grade Systemic Inflammation Change Tumor Markers Levels in Patients on Hemodialysis?

Introduction: End-stage renal disease (ESRD) patients are known to have a high risk of developing cancer-related inflammation. Elevated serum levels of tumor markers in ESRD/hemodialysis patients makes analysis and interpretation difficult. Aim: To verify the possible relationship between chronic low-grade systemic inflammation serum levels determined by C-reactive protein (CRP) and the tumor biomarkers in patients on hemodialysis. Materials and methods: A prospective study of prevalence was conducted in the Hemodialysis Sector of the University Hospital of the University of Brasília between July 2016 and December 2016 in men aged 18 to 60 years without clinically detectable cancer. We assessed inflammation by serum high-sensitivity CRP test (hs-CRP) and serum tumor in the case groups and controls. The hemodialysis group was split into two subgroups: group 1: patients with inflammation (CRP > 5 mg/L, n=27), and group 2: patients without inflammation (CRP ≤5 mg/L, n=33). Results: There was no significant difference in age mean levels between case groups and controls (44.00±08.00 vs. 41.00±07.00, p=0.08). There was no difference or correlation (p>0.05) between tumor markers levels and patients with and without inflammation. Conclusions: The results of this study suggest that chronic low-grade systemic inflammation defined by C-reactive protein serum levels does not promote elevated serum PSA levels in chronic hemodialysis patients.


INTRODUCTION
Different studies in the past have shown that the risk of cancer development is increased in end-stage renal disease (ESRD) patients, whether on dialysis or not, when compared with the general population. 1,2 Lin et al. 2 checked in dialysis (hemodialysis and peritoneal dialysis) group a significantly higher 7-year cancer incidence rate (6.4%) than did the control group and by multivariable analyses confirmed the association between long-term dialysis and cancer and concluded that dialysis is associated with a higher risk of cancer in patients with ESRD. Identification of specific cancer risks is crucial in the cancer screening of these patients.
On the other hand, the presence of elevated serum levels of tumor markers in ESRD/ hemodialysis patients makes analysis and interpretation difficult. Considering the real risk of cancer and the false positive in that can raise the concentration of tumor markers in the absence of cancer by several causes with methodological, physiological or pathological. 3 False positives with a methodological origin include water permeability (low flux or high flux) and principle of hemodialysis involves the clearance of solutes across a semi-permeable membrane (diffusion, convection, adsorption and ultrafiltration mechanisms). 3 False positives with a pathological origin include alterations that increase marker release due to the alterations that reduce its elimination (such as kidney or liver failure), necrosis or inflammation. 3 The chronic low-grade systemic inflammation (CLGSI) is regarded as a common comorbid condition in chronic kidney disease (CKD) 4 and a confounding factor in the interpretation and analysis of elevated serum levels of tumor markers of ESRD/hemodialysis patients by the prevalence of high inflammatory factor in this group of patients. 5 Low-grade chronic systemic inflammation in CKD may cause elevated levels of tumor markers due to a likely link between chronic inflammation and development of neoplasia. 6 In this context, it was decided to verify the possible relationship between CLGSI serum level determined by C-reactive protein (CRP) and tumor biomarkers in patients on haemodialysis.

MATERIALS AND METHODS
A prospective study of prevalence realized in the Hemodialysis Sector of the University Hospital of the University of Brasília, between July 2016 and December 2016, after approval by the Research Ethics Committee of the Faculty of Health Sciences of the University of Brasília (No 53172316.9.0000.0030). We assessed inflammation via serum high-sensitivity CRP (hs-CRP) measurements in the group case and control. Patients in hemodialysis group were classified into two subgroups: group 1: with inflammation (CRP > 5 mg/L, n=27), group 2: without inflammation (CRP ≤5 mg/L, n=33) based on the recommendation of the National Kidney Foundation. 7 The control group (group 3, n=20) were subjects from the health promotion general outpatient clinic of the same hospital who had normal glomerular filtration rate and 90 ml/min/1.73 m 2 . CRP value of ≤1 mg/L is considered normal for healthy patients. 8 The patients included in the study were aged 18-60 years, without clinically detectable cancer. The case group was undergoing hemodialysis for >6 months, received high-flux hemodialysis three 4-h dialysis weekly sessions, using fistula as vascular access.
Exclusion criteria were chronic inflammation due to positive serology for hepatitis B, C, or HIV. Patients with acute or chronic liver disease, rectal examination in the previous week, prostate biopsy in the previous 4 months, cystoscopy, history of urinary tract infection, clinical signs of acute or chronic infection/inflammation, vascular access infection, leukocytosis, fever, or hypoproteinemia were not included in the study.
Blood samples were collected for analysis at the same time, between 8:00 a.m. and 10:00 a.m. in the clinical laboratory of the same hospital. The samples were taken from the arteriovenous fistula immediately before the first weekly hemodialysis session in the case group and on a previously scheduled day in the control group. Five millilitres of blood were collected via arm venoclysis without anticoagulant administration. The blood was centrifuged at 3500×g for 20 min, and the supernatant was collected in a centrifuge tube and maintained at -20°C until tumor markers (alpha-fetoprotein, cancer antigen 19-9, carcinoembryonic antigen, lactate dehydrogenase) and CRP measurements.
Serum CRP levels were measured using turbidimetry in the automatic analyzer BN II (Dade Berhing, kit Dade Berhing, USA). Tumor markers levels were measured via enzyme immunochemoluminescence by using automatic analyzer Immulite 2000/Siemens. Specific kits in addition to calibrators and controls recommended by the manufacturer were used to quantify measurements. All statistical analyses were performed using SPSS® for Windows, version 24.0.
After analyzing the sample distribution by using the Shapiro-Wilk or Kolmogorov-Smirnov normality test, differences between three independent quantitative variables were evaluated using the Kruskal-Wallis test when abnormal distribution curve was presented. Those between three independent quantitative variables with normal distribution curve were evaluated using one-way ANOVA test. Statistical significance was set at p<0.05 to reject the null hypothesis.

RESULTS
There was no significant difference in age mean levels between case and control groups (44.00±08.00 vs. 41.00±07.00, p=0.08). There was no difference (p>0.05) between tumor markers levels of patients with inflammation (CRP > 5 mg/L) and patients without inflammation (CRP ≤5 mg/L). CRP serum levels have no correlation with tumor markers (p>0.05) ( Table 1).

DISCUSSION
The present study has its relevance because it is one of the first to verify the possible effect of chronic systemic inflammation prevalent in these patients on the biomarkers levels, helping in the analysis and interpretation of these levels above normal limits.
Despite the evidence key component of cancer-related inflammation 6,9 , this seems to be ignored in most biomar-ker studies with confounding conditions. The prevalence of signs of inflammation are inversely related to the level of kidney function and positively associated with the magnitude of proteinuria. 4,10 The etiology of inflammation in CKD is multifactorial, triggering orchestra action to produce key mediators molecules (cytokines and acute-phase proteins) of the inflammatory process as well as markers of inflammation. 4 The similar age between the groups studied (p>0.05) ensures greater reliability in the interpretation of the results and comparative statistical analysis of the impact of age on outcome variables.
Our results show that CRP serum levels do not significantly influence to increase (comparative evaluation (p>0.05) ( Table 2) or maintain correlation (correlational evaluation, p>0.05) ( Table 1) with the tumor markers analyzed in patients undergoing hemodialysis. These results can be explained, in part, by the high-flux type of membranes used in the hemodialysis. These membranes are known to be capable of removing moderate-size molecules between 10000 to 15000 Dalton, including many of the inflammatory proteins, tumor markers, ß₂ macroglobulin, lipoproteins and a wide spectrum of uremic toxins. 11,12 As result, in theory, high-flow hemodialysis would have a direct effect on the inflammatory cytokines and/or serum levels of tumor markers.
In the absence of cancer, the increase in the plasma concentration of a tumour marker above the upper reference limit established for healthy individuals is considered a false positive. There are different types of false positives which can be classified as methodological, physiological or pathological. 3 Methodological false positives are attributed to the measuring system; they arise due to the lack of specificity of the antibody, crossed reactions with other molecules, or due to the presence of heterophil antibodies. 3 The pathological false positives include alterations that increase marker release due to necrosis or inflammation, and the alterations that reduce its elimination (such as kidney or liver failure). 13 The physiopathological processes that link the increase of plasma concentrations of tumor markers to CLGSI is not understood; however, it has been hypothesized that inflam-mation may play a role in cancer development or cancer may cause an inflammatory response and influence measured biomarker levels. 14 This vicious circle of events generates cancer-promoting local and systemic inflammatory microenvironment 14,15 and may be a confounding factor for analysis and interpretation of the serum levels of tumor markers. 16 For Thompson et al. 14 it is this chronically inflamed state, a pro-inflammatory state sustains the release of cytokines and chemokines with the capability of causing progressive alterations in the cellular and molecular composition of the microenvironment. This leads to elevated levels of promutagenic reactive oxygen and reactive nitrogen species, alterations in the vasculature (e.g. vascular hyperpermeability, neovascularization, and angiogenesis), disturbances in mitochondrial function, and, most importantly, the disruption of normal cell-cell signalling/cross-talk such as recruitment of macrophages with suppressive function to disable T cell-mediated tumor immunity.
Alternatively, molecular, cellular and tissue alterations caused by the chronic inflammation can induce the synthesis and secretion of tumor markers. 13 The limitations of the present study are the use of CRP as a non-specific marker of inflammation and the reduced number of sample and tumor markers. However, the CRP is a serological marker of inflammation that can be used to investigate the association between inflammation and risk of cancer practically at low cost. 17

CONCLUSIONS
The results of this study suggest that the chronic low-grade systemic inflammation defined by C-reactive protein serum levels does not promote elevated serum PSA levels in chronic hemodialysis patients.