Research on changes of malondialdehyde content in patients with colon cancer before and after radical surgery

ls, free radicals. Studies also show that increased intestinal oxidation is associated with risk factors for colon cancer such as chronic ulcerative colitis, obesity, lack of physical exercise, and a diet high in red meat, smoking and alcohol abuse. Studies on molecular biology show that oxidative stress may affect mutations of the genes K-Ras, p53, APC or MMR (DNA mismatch repair gene), these genes play an important role in leading to colon cancer. In addition, clinical studies also indicate that the level of oxidative stress is related to the factors expressing the progression of cancer, such as lymph node metastasis, venous invasion, disease stage... 1.3. Role of oxidative stress in recurrence of colon cancer after surgery There are many factors that affect the recurrence of colon cancer after surgery have been identified such as: biological characteristics and histopathology of tumors, postoperative chemical treatment, surgical techniques, in which the stage of the disease is the most important factor determining the postoperative survival rate and also the factor the most affecting the recurrence rate after surgery. Currently, there are many evidences showing that surgery process itself also contributes to postoperative outcomes of cancer treatment. Surgery, even though it works for therapeutic purposes, is still a traumatic impact on the body and is also an exogenous cause producing many free radicals and oxidative stress because trauma in surgery causes the body's “stress”, which is called surgical stress, leads to an increase in free radicals through activating ROS-producing enzymes such as XO (xanthine oxidase), Cox (Cyclooxygenase) and especially NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) or also called Nox. Abdominal surgery also increases free radicals and oxidative stress through the mechanism of ischemia-reperfusion injury due to the effect of increasing the abdominal pressure in laparoscopic surgery, exposure to operating room air in open surgery and mechanical operations on the intestine. Recent studies have also demonstrated that free radicals containing active oxygen species (ROS) and redox signaling appear when surgery plays a role in promoting invasive and metastatic growth of mass tumor cells left after surgery to treat colon cancer. The process of cancer cells left after surgery develops into metastases in the new organ, the following phenomena are required: invasive cancer cells (Invadopodia formation), escape from the capillaries to adhere to new tissues, new organs (Adhesion), Angiogenesis, and uncontrolled proliferation cells. ROS generated during the surgery play a role of motivating, facilitating these phenomena to occur. 1.4. Biological indicators (biomarkers) to assess oxidative stress in surgery There are many methods to assess the body's oxidative stress. The direct method is to quantify ROS and the indirect method is to assess through measurement of stable metabolites of ROS or products of ROS process interacting with biological molecules such as lipids, proteins, DNA. In the body, ROS exists in a very short time and their concentration is very low. To determine ROS, it requires modern and expensive techniques and equipment, so clinical studies often use indirect methods to assess oxidative stress. The oxidation of biological molecules produces a wide range of products that can be used as biological indicators to assess oxidative stress such as: Carbonyl proteins (Product of protein oxidation process), 8-OHdG (Product of DNA oxidation process)... However, the product which is used the most in studies to assess oxidative stress in general and in abdominal surgery in particular is Malondialdehy (MDA) of lipid peroxidation. According to Pappas-Gogos G. (2013), the lipids of cell membranes are the molecules involved the earliest in reacting with free radicals, while oxidation of proteins and DNA occurs at a later time. In addition, MDA is the most commonly used in many studies because it is easily quantified by quantification of MDA derivatives with TBA (Thiobacbituric Acid), and this quantification of derivatives can use a very simple, low cost, convenient spectroscopic method, which can be done on many different biological samples. 1.5. Studies on Malondialdehyde in colon cancer patients in the world and in Vietnam MDA, an index commonly used to assess oxidative stress, has also been used in many studies to evaluate on colon cancer patients. MDA was quantified in serum, plasma, red blood cells, urine and colon tissue to make comparisons between the two groups: 1 group is colon cancer patients, 1 reference group is healthy persons, as well as explore the relationship between MDA index and pathological factors in colon cancer. The changes in oxidative stress have been studied after surgery for lung cancer, breast cancer and also studied after surgery for colon cancer. All studies have shown that oxidative stress occurs right after surgery for colon cancer and tends to decrease over time after surgery, however, there is a difference among studies in time and evaluation index. Studies have also explored the role of laparoscopic surgery, of breathing with high oxygen levels or blood transfusion in surgery to oxidative stress in surgery, but the effect of surgery duration or the relationship between postoperative oxidative stress and surgical outcomes has not been mentioned in any study. In Vietnam, there have been no studies finding out about MDA content before surgery and the change of MDA after radical surgery to treat colon cancer. CHAPTER 2 SUBJECTS AND RESEARCH METHODOLOGY 2.1. Research subjects A total of 74 patients who were diagnosed with colon cancer, underwent inpatient treatment at the Department of Abdominal Surgery - Military Hospital 103 and was carried out radical surgical treatment from March 2015 to January 2017 (serve for target 1). Of which, 60 patients had enough test results of red blood cell MDA index at 4 times before and after radical surgery (serve for target 2). 2.1.1. Selection criteria - Criteria for identifying colon tumors: Applying the International Classification of Diseases for Oncology, 3rd Edition of the World Health Organization, the position of the tumor in the colon cancer determined from cecum to the end of the sigma colon, above the rectosigmoid junction. In this study, the tumor was identified as of the colon, with the tumor position 15cm away from the anal margin. - Colon cancer patients at stages I, II and III. The anatomical result on the tumor cell morphology is adenocarcinoma. - Carry out radical surgery to remove the tumor in the colon, with a surgery minutes describing in details the lesions and techniques. - Follow the procedure of treatment, nursing before, during and after surgery, have complete medical records and voluntarily agree to give disease samples for research. 2.1.2. Exclusion criteria - Recurrent colon cancer or secondary colon due to that cancer from other organs metastasize. - Colon cancer has been treated with chemicals and radiation therapy before surgery. - Colon cancer must undergo emergency surgery. - Colon cancer associated with other diseases combined (diabetes, cardiovascular disease, systemic disease ), smoking history, BMI >30. 2.2. Research methodology 2.2.1. Research design Prospective study, no-control cross sectional description, with before-after comparison. 2.2.2. Sample size: With the main objective of comparing MDA content between tumor tissue with normal colon tissue and investigating the change of peripheral erythrocyte MDA before and after the surgery, we applied the formula to calculate the minimum sample size following Miot H. A. (2011). Of which n: sample size, Zα/2: value of error α, Zβ: value of error β, in this research (Z α/2 + Zß)² = 10.5 with α is 0.05, ß is 0.1. Sa and Sb: standard deviation of the variable in each group, d: minimum difference between the mean values. Referring to research of Upadhya S. and CS (2004) (a study comparing erythrocyte MDA content between colorectal cancer groups and healthy people), with Sa = 3.4, Sb = 5,9; d = 2,9 (8,3-5,4). Replace the above formula: n = 57.89. Therefore, the minimum expected sample size is 58 patients. 2.2.4. Method to determine MDA content 2.2.4.1. Principle of the MDA quantitative method The principle of the method is based on the chemical reaction of MDA molecule with thiobarbituric acid (TBA) (also called TBA test method). MDA reacts with TBA in proportion 1:2 to create MDA-(TBA)2 complex which has a characteristic pink color, the reaction takes place at temperature 98oC, appropriate pH from 2-3. The MDA-(TBA)2 complex was measured at a maximum absorption wavelength of 535 nm using a spectrophotometer. From this absorbance value, the corresponding MDA amount in the reaction solution will be determined, thereby quantifying the MDA content in the sample. 2.2.4.2. Measurement of MDA for tissue samples Tissue samples of patients with colon cancer for MDA index determination are provided by the Department of Anatomic Pathology, Military Hospital 103. The colon segment containing the tumor after being removed from the patient via surgery will be cold stored in an ice box (without formalin) and taken to the Department of Anatomic Pathology. Here, tissue samples are taken at two locations: diseased tissue is taken at the tumor and normal tissue is taken at a location at least 5cm from the tumor edge. The tissue samples were then stored at minus 1960C with liquid nitrogen before being taken to oxidative stress testing. The method of quantifying MDA on tissue samples was carried out based on the TBA test method, developed by Uchiyama M. (1978). 2.2.4.3. Measurement of Red blood cell MDA Blood samples of patients with colon cancer for MDA index determination are taken from peripheral veins. Next, the blood sample was centrifuged at a speed of 3000 rpm for about 10 minutes to separate plasma and red blood cells. The red blood cells were stored at 40C before being analyzed for MDA index. The determination method is the same as for the tissue sample, but because it is difficult to measure the weight of the erythrocyte membrane (or red blood cell membrane) in the blood sample, therefore to determine the MDA content on erythrocyte membrane, it is necessary to determine the protein content of the erythrocyte membrane and MDA value calculated on 1 mg of red blood protein. 2.2.5. Research criteria 2.2.5.1. Criterion on characteristics of the research group Including criteria: age, gender, body mass index, anemia, neutrophil/lymphocyte ratio (NLR), CEA concentration, characteristics of radical surgery, postoperative tumor anatomic pathology. Criterion on early results after surgery Including criteria: complications in surgery, death - complications after surgery, time of postoperative ileus, number of fever days after surgery, time of hospitalization after surgery. 2.2.5.3. Criterion on the MDA content Including 3 criteria: MDA content in tumor tissue, MDA content in colon normal tissue (taken from tumor edge > 5cm) cells before surgery taken at the time before surgery. The MDA concentration in the tissue was measured in µg/g sample, the MDA concentration of peripheral red blood cells before surgery was measured in µg/mg Protein. 2.2.5.4. Criterion on changes of erythrocyte MDA content after surgery Peripheral erythrocyte MDA at 3 times: 1 day after surgery (after 24 hours), 3 days after surgery (after 72 hours) and 7 days after surgery (after 168 hours). The MDA content of blood samples after surgery was also measured in µg/mg Protein. 2.2.5. Data processing method Comparing MDA values at four points before and after surgery using Friedman test, comparing MDA values between two points or between two tissue positions in the colon using the paired samples Wilcoxon test. The statistical analysis was performed by SPSS 20.0, the difference was statistically significant if p <0.05. 2.3. Ethical issues in research All patients voluntarily participate in the research and the research does not affect the quality of treatment, does not cause invasion, damage to patients. For MDA testing in this study, patients do not have to pay. CHAPTER 3 RESEARCH RESULTS 3.1. Characteristics of the research group and early results after surgery 3.1.1. Age, gender, and body mass index Average age: 59.8 ± 11.9 (29 - 87). Male: 42 patients (56.8%). Female: 32 patients (43.2%). Male/Female ratio: 1.31/1. Weight: normal 62.2%; underweight 21.6%; overweight 16.2%; No patients had obesity. 3.1.2. Blood tests before surgery Anemia: 39 patients (52.7%). NLR ≥ 4: 20 patients (27%). CEA concentration before surgery > 10 ng/ml: 19 patients (25.7%). 3.1.3. Anatomical Pathology after surgery - Right colon cancer: 31 patients (41.9%), Left colon cancer: 43 patients (58.1%), sigma colon cancer accounts for the highest rate: 44.6%. The average tumor size is 6.1 ± 2.4 cm (2 - 12cm). There were 19 patients (25.6%) having tumor adhering to adjacent organs. - The average number of lymph nodes dredged in one patient was 15.5 ± 7.7 nodes (2 - 38). Number of patients with lymph nodes having ≥ 12 lymph nodes: 47 patients (63.5%). The average length of the removed colon segment is 31.6 ± 17.1 (12 - 85cm). - Patients in Stage I, II, III correspondingly were 11 patients (14.9%), 32 patients (43.2%) and 31 patients (41.9%). The rate of patients with lymph node metastasis: 41.9%, invasion T4: 33 patients (44.6%). 3.1.4. Radical surgery - Laparoscopic surgery: 42 patients and open surgery: 32 patients, Colostomy: 7 patients (9.5%). Expanding surgery: 14 patients (18.9%), of which 5 patients with total colectomy. Most patients with expanding surgery were those who underwent open surgery (13/14). - The average length of time required to perform a surgical procedure: 134.6 ± 38.7 minutes (75 - 270). 3.1.5. Early results after radical surgery - No complications in surgery, death after surgery: 1 patient. The rate of complications after surgery: 4 patients (5.4%). - Average postoperative ileus time: 81.1 ± 22.2 hours (34 - 130 hours). Average number of fever days after surgery: 1.7 ± 1.2 days (1 - 7 days). Average length of hospital stay after surgery: 9.5 ± 2.9 days (6 - 21 days). 3.2. MDA content result in patients with colon cancer before radical surgery 3.2.1. MDA content in tumors tissue, normal tissue of colon and peripheral red blood cells Chart 3.3. Comparison of MDA content of tumor tissue and normal colon tissue Table 3.12. MDA content in tumor tissue, colon normal tissue and peripheral red blood cells MDA n Min Max Average Median p* Normal tissue (µg/g sample) 74 0.58 3.40 1.52 ± 0.56 1.40 0.005 Tumor tissue (µg/g sample) 74 0.55 4.35 1.73 ± 0.76 1.52 Red blood cells (µg/mg Protein) 74 0.020 0,.624 0.167 ± 0.10 0.142 *Wilcoxon test - MDA content of tumor tissue was higher than that of normal colon tissue with p <0.05. There was a correlation between MDA content of tumor tissue and normal tissue with Spearman correlation coefficient: rs = 0.549; p <0.001. - There was no correlation between red blood cell MDA and MDA content of tumor tissue and normal tissue. 3.2.2. Analysis of tumor tissue MDA content based on a number of clinical and pathological factors Table 3.19. MDA content of tumor tissue according to the stage disease Element n Average MDA content (µg/g sample) p* Disease stage I 11 1.68 ± 0.61 0.890 II 32 1.63 ± 0.59 III 31 1.85 ± 0.94 Total 74 1.73 ± 0.76 * Mann-Whitney test Table 3.21. MDA content of tumor tissue according to tumor position. Element n Average MDA content (µg/g sample) p* Tumor position Colon P 31 1.92 ± 0.71 0.017 Colon T 43 1.59 ± 0.77 Total 74 1.73 ± 0.76 * Mann-Whitney test - The average MDA content in tumor tissue of the right colon cancer group higher than that of the left colon cancer group has statistical significance with p <0.05. - Analyze the tumor tissue MDA content by elements: Age ( 10 ng/ml), Disease stage (I, II, III), tumor size ( 0.05. 3.2.3. Analysis of peripheral red blood cell MDA content before surgery according to some clinical and pathological factors Table 3.27. Red blood cell MDA content before surgery according to disease stage Element n Average MDA content (µg/mg Protein) p* Disease stage I 11 0.232 ± 0.10 0.049 II 32 0.154 ± 0.10 III 31 0.158 ± 0.08 Total 74 0.167 ± 0.10 *Kruskal-Wallis test Table 3.28. Red blood cell MDA content before surgery according to tumor size Element n Average MDA content (µg/mg Protein) p* Tumor size < 6cm 36 0.196 ± 0.11 0.017 ≥ 6cm 38 0.140 ± 0.07 Total 74 0.167 ± 0.10 * Mann-Whitney test - The preoperative RBC MDA content in the patient group of stage I is higher than that in patient groups of stages II and III; in the group with tumor size < 6cm higher than the group with tumor size ≥ 6cm, it is statistically significant with p <0.05. - Analyze the preoperative RBC MDA content by elements: Age ( 10 ng/ml), tumor position (right and left colon cancer), realize the same result: the preoperative RBC MDA content by the above group of elements had no difference with p> 0.05. 3.3. Changes of RBC MDA content in colon cancer patients after radical surgery 3.3.1. Red blood cell MDA content at the point before and after surgery Table 3.30. Red blood cell MDA content at the points MDA content (µg/mg Protein) n min max Average Median Before surgery 60 0.045 0.624 0.168 ± 0.10 0.145 1 day after surgery 60 0.070 0.789 0.217 ± 0.13 0.186 3 days after surgery 60 0.055 0.557 0.190 ± 0.11 0.147 7 days after surgery 60 0.034 0.726 0.179 ± 0.12 0.152 p* 0.001 * Friedman test Table 3.31. Compare the RBC MDA content paring at the points p* (n=60) MDA before surgery (0.168 ± 0.10) MDA day 1 (0.217 ± 0.13) MDA day 3 (0.190 ± 0.11) MDA day 7 (0.179 ± 0.12) MDA before surgery (0,168 ± 0.10) 1 MDA day 1 (0.217 ± 0.13) 0.001 1 MDA day 3 (0.190 ± 0.11) 0.795 0.020 1 MDA day 7 (0.179 ± 0.12) 0.985 0.003 0.942 1 * Wilcoxon test Chart 3.5. Comparing RBC MDA content at different points - Immediately after surgery, the MDA content of red blood cells increases and then gradually decreases. At the point 1 day after surgery, the RBC MDA content reached the highest, it was higher than other times and had statistical significance with p 0.05 (Willcoxon test). 3.3.2. Changes in MDA content of red blood cells after surgery according to surgical characteristics Table 3.32. RBC MDA content at different points according to surgery method MDA content (µg/mg Protein) n Surgery method p* Laparoscopy (n=34) Open surgery (n=26) Before surgery 60 0.174 ± 0.11 0.161 ± 0.09 0.536 1 day after surgery 60 0.233 ± 0.13 0.197 ± 0.11 0.187 3 days after surgery 60 0.200 ± 0.12 0.177 ± 0.11 0.408 7 days after surgery 60 0.200 ± 0.14 0.151 ± 0.09 0.072 p** 0.005 0.039 * Mann-Whitney test ** Friedman test Table 3.35. RBC MDA content at different points according to surgery duration MDA content (µg/mg Protein) n Surgery duration (SD) p* < 130 mins (n=30) ≥ 130 mins (n=30) Before surgery 60 0.186 ± 0.12 0.151 ± 0.07 0.387 1 day after surgery 60 0.227 ± 0.15 0.208 ± 0.09 0.912 3 days after surgery 60 0.195 ± 0.14 0.184 ± 0.09 0.802 7 days after surgery 60 0.192 ± 0.15 0.165 ± 0.07 0.756 p** 0.014 0.002 * Mann-Whitney test ** Friedman test - Comparing the pairing of points by the laparoscopic group and open surgery group indicated that the RBC MDA content in the laparoscopic group at the point of 3 days after surgery lower that of 1 day after surgery had statistical significance (p 0.05). - Comparing the pairing of points by surgery duration: SD 0.05; meanwhile, in the group with SD ≥ 130 mins, the RBC MDA content of 1 day after surgery and 3 days after surgery were higher than that before surgery, it was statistical significant with p <0.05. 3.3.3. Changes in RBC MDA after surgery related to early postoperative results Table 3.40. RBC MDA content at different points according to postoperative ileus time MDA content (µg/mg Protein) n postoperative ileus time (PIT) p* < 72 hrs (n=25) ≥ 72 hrs (n=35) Before surgery 60 0.147 ± 0.07 0.184 ± 0.11 0.393 1 day after surgery 60 0.183 ± 0.07 0.242 ± 0.15 0.304 3 days after surgery 60 0.149 ± 0.06 0.219 ± 0.14 0.078 7 days after surgery 60 0.154 ± 0.07 0.196 ± 0.14 0.664 p** 0.004 0.039 * Mann-Whitney test ** Friedman test - Comparing the pairing of points by group: PIT 0.05). Table 3.43. RBC MDA content at different points according to number of fever days after surgery MDA Content (µg/mg Protein) n Number of fever days after surgery (NFDAS) p* < 3 days (n=48) ≥ 3 days (n=12) Before surgery 60 0.159 ± 0.10 0.205 ± 0.09 0.093 1 day after surgery 60 0.207 ± 0.13 0.257 ± 0.07 0.026 3 days after surgery 60 0.182 ± 0.11 0.224 ± 0.13 0.139 7 days after surgery 60 0.164 ± 0.10 0.238 ± 0.18 0.177 p** 0.007 0.04 * Mann-Whitney test ** Friedman test - Comparing the pairing of points by group: NFDAS 0.05). Table 3.46. RBC MDA content at different points according to length of hospital stay MDA content (µg/mg Protein) n Length of hospital stay after surgery (LHSAS) p* < 10 days (n=35) ≥10 days(n=25) Before surgery 60 0.139 ± 0.06 0.210 ± 0.13 0.067 1 day after surgery 60 0.188 ± 0.08 0.258 ± 0.16 0.144 3 days after surgery 60 0.168 ± 0.09 0.221 ± 0.14 0.175 7 days after surgery 60 0.157 ± 0.06 0.209 ± 0.17 0.893 p** 0.015 0.017 * Mann-Whitney test ** Friedman test - Comparing the pairing of points by group: LHSAS 0.05). DISCUSSION 4.1. Characteristics of research group and early results after surgery In this study, only patients in stage I, II and III who underwent radical surgery were included, we excluded cases of incomplete tumor resection and had distant metastases or had complication required for an emergency surgery. However, through the obtained results, we realized that the characteristics of the research group such as: average age, age group distribution, male/female ratio, anemia, preoperative CEA concentration, tumor histopathology as well as the early results after surgery had no difference with the previous domestic studies on colon cancer. The neutrophil to lymphocyte ratio (NLR) in peripheral blood before surgery is an index reflecting inflammatory response, recently concerned by many researchers. In this research, tumors invadin adjacent organs often have a NLR ≥ 4, indicating that the tumor has caused a systemic inflammatory response. 4.2. Characteristics of MDA content before surgery in colon cancer patients being treated with radical surgery 4.2.1. Compare MDA content in tumor tissue, normal colon tissue and peripheral red blood cells To explore the characteristics of MDA content before surgery in patients with colon cancer who were treated with radical surgery, we conducted a quantification of MDA in 3 biological samples: tumor tissue, normal colon tissue taken at least 5cm far from the tumor and peripheral red blood cells. Similar to the results of Hendrickse C. (1994), Skrzydlewska E. (2005), our research results show that the MDA content in tumor tissue is higher than that in normal colon tissue. Especially, in the study of Veljković A. (2016), the MDA concentrations in tumor tissue and surrounding tissue are higher than healthy tissue far from the tumor. The increase in MDA levels in the diseased tissue in this study once again proves that although the exact mechanism of oxidative stress in colon cancer is not fully known, lipid peroxidation takes place more strongly in cancerous cells compared to healthy colon cells in patients with colon cancer. In addition, the results of analyzing the correlation between MDA content of tumors and MDA of normal tissue indicated that they have a relatively close positive correlation, and that oxidative stress is not only increasing in cancerous tissue but also appears in healthy tissue of colon cancer patients. The analysis result does not find out the correlation between MDA in red blood cell and MDA in tumor tissue as well as normal colon tissue, and it indicates that the typical biological activity of colon cancer which may lead to oxidative stress in the tumor tissue has distinct characteristics compared to that in blood tissue. 4.2.2. Factors affecting MDA content in tumor tissue and MDA in peripheral red blood cells Surveying some clinical and subclinical factors such as age, gender, anemia, leukocyte ratio, CEA concentration, tumor anatomic pathology, research results show that among the above factors, only some tumor characteristics