VIETNAM MILITARY MEDICAL UNIVERSITY======== PHAM MANH CUONG RESEARCH ON CHANGES OF MALONDIALDEHYDE CONTENT IN PATIENTS WITH COLON CANCER BEFORE AND AFTER RADICAL SURGERY Major : Gastroi
Trang 1VIETNAM MILITARY MEDICAL UNIVERSITY
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PHAM MANH CUONG
RESEARCH ON CHANGES OF MALONDIALDEHYDE CONTENT IN PATIENTS WITH COLON CANCER BEFORE AND AFTER RADICAL SURGERY
Major : Gastrointestinal surgery Code : 9 72 01 04
SUMMARY OF MEDICAL DOCTORAL THESIS
HANOI - 2019
Trang 21 Ass Prof Nguyen Van Xuyen
2 Ass Prof Trinh Hong Thai
Reviewer 1: Ass Prof Nguyen Xuan Hung
Reviewer 2: Prof Ha Van Quyet
Reviewer 3: Ass Prof Nguyen Anh Tuan
The thesis will be defended in front of the Reviewing Council at the University level at:
Trang 31 Pham Manh Cuong, Nguyen Van Xuyen (2018) Understanding
factors related to length of the colon section removed afterradical surgery for colon cancer treatment at Military Hospital
103 Journal of Military Pharmaco - Medicine, 43 (special
subject of abdominal surgery): 44-50
2 Pham Manh Cuong, Nguyen Van Xuyen, Trinh Hong Thai
(2019) Investigation on changes in the erythrocyteMalondialdehyde value in patients with colon cancer after
radical surgery Journal of military pharmaco - medicine, 44(2):
213-219
3 Pham Manh Cuong, Nguyen Van Xuyen, Trinh Hong Thai
(2019) The comparison of oxidative stress between tumourtissue and healthy colon tissue using the Malondialdehyde index
in colon cancer patients undergoing radical surgery at 103
Military Hospital VietNam Medical Journal, 481(1): 71-75.
Trang 4Currently, the strong development of researches on themechanism of action and consequences of free radicals, as well asoxidative stress on the body has provided evidence showing thatoxidative stress and Oxygen-free radicals are also a pathologicalfactor involved in the development of colon cancer
In particular, the results of many studies show that oxidative stress and the presence of oxygen-free radicals, produced during
surgery removing colon cancer, also play an important role in relapseand metastasis after surgery Therefore, the study of changes inoxidative stress after surgery is currently of interest to manyresearchers There are many indices to assess oxidative stress status,but in the studies, the most widely and commonly applied index toindirectly assess oxidative stress in general and in abdominal surgery
in particular is Malondialdehyde (MDA), a product of lipidperoxidation
In Vietnam, not many studies mentioned the issue of oxidativestress in colon cancer and exploring the changes in MDA contentafter radical surgery for colon cancer treatment Therefore, we
conducted the topic “Research on the changes in Malondialdehyde content in patients with colon cancer before and after radical surgery” with the following goals:
1 Study the Malondialdehyde content in patients with colon cancer who underwent radical surgery treatment.
2 Evaluate the changes in red blood cell Malondialdehyde content of patients with colon cancer after radical surgery.
Layout of the thesis
The thesis consists of 124 pages, including the following parts:
Trang 5Background (2 pages), Overview (38 pages), Subjects and research methodology (20 pages), Results (32 pages), Discussion (30 pages),Conclusion (2 pages) The thesis has 56 tables, 11 figures, 11 charts,
1 diagram, 173 references including 11 Vietnamese documents, 162English documents, and 53 documents within 5 years
CHAPTER 1 OVERVIEW 1.1 Colon cancer and radical surgery to treat colon cancer
Radical surgery is the main method in colon cancer treatment,chemicals and radiation therapy are complementary method tosurgery Currently, there has not yet been a specific and consensusstandard to precisely determine the radical surgery for colon cancer;however, to ensure the treatment in cancer surgery, radical surgery isrequired to comply with the following requirements
* Diagnosis of the disease stage before surgery
Examining the entire colon (with one or more tumors, polypscombined), diagnosing the disease stages (with lymph nodemetastases, distant metastases), assessing the local invasion of thetumor before surgery exactly are essential to plan the radical surgeryfor colon cancer
* Vertical colon resection extent
For colon cancer, a length of 5cm is defined as no more cancerorganization and will not cause recurrence at the junction In surgery,the actual length of the colon segment removed will be determined bythe removal extent of the colon arteries, parallel to the lymphaticdrainage This length may have to be extended, depending on theextent of lymph node dredging
Trang 6* Resection extent of invaded organs
During surgery, it is difficult to identify if an organ or organization that is attached to the tumor is due to inflammation or invasive coloncancer Therefore, when the tumor invades a nearby organ, in surgery itshould be removed the tumor together with the invaded organ in anintegral block (en bloc resection)
* Extent of lymph node dredging
At the time of surgery, according to many studies up to 50% ofcolon cancer possibly has regional lymph node metastases, so thelymph node dredging is an indispensable part and lymph nodedredging must be done thoroughly
1.2 Oxidative stress and the role of oxidative stress in colon cancer
In the body, free radicals are frequently created, due to containingoxygen, they are also called reactive oxygen species (ROS), and theyare always in balance with the body's antioxidant system Oxidativestress is the state where the formation of ROS is beyond the control ofthe antioxidant system, the result is that ROS will attack biologicalmolecules such as: Lipids (lipid peroxidation), Protein ( proteinoxidation), Nucleic acid (DNA oxidation) leading to changes inbiological molecules, producing a number of toxic products thatdamage cells, tissues and result in abnormal activities of the body Inaddition, through ROS, oxidative stress also affects cell signalingpathways, causing false information leading to abnormal development
of cells such as uncontrolled cancer cell proliferation
Similar to the respiratory tract, the intestinal tract are the organ that most exposed to the environment through the food digestion process In particular, the bacteria are concentrated much in the colon
Trang 7and very little in the small intestine, so the colon mucosa cells are constantly exposed to chemicals, free radicals Studies also show thatincreased intestinal oxidation is associated with risk factors for colon cancer such as chronic ulcerative colitis, obesity, lack of physicalexercise, and a diet high in red meat, smoking and alcohol abuse.Studies on molecular biology show that oxidative stress may affectmutations of the genes K-Ras, p53, APC or MMR (DNA mismatchrepair gene), these genes play an important role in leading to coloncancer In addition, clinical studies also indicate that the level ofoxidative stress is related to the factors expressing the progression ofcancer, 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 cancerafter surgery have been identified such as: biological characteristicsand 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 alsothe factor the most affecting the recurrence rate after surgery.Currently, there are many evidences showing that surgery processitself 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
Trang 8adenine 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 mechanicaloperations on the intestine
Recent studies have also demonstrated that free radicalscontaining active oxygen species (ROS) and redox signaling appearwhen surgery plays a role in promoting invasive and metastaticgrowth 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: invasivecancer cells (Invadopodia formation), escape from the capillaries toadhere to new tissues, new organs (Adhesion), Angiogenesis, anduncontrolled proliferation cells ROS generated during the surgeryplay 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 toassess 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 theirconcentration is very low To determine ROS, it requires modern andexpensive techniques and equipment, so clinical studies often useindirect 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
Trang 9stress 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 inreacting with free radicals, while oxidation of proteins and DNAoccurs at a later time
In addition, MDA is the most commonly used in many studiesbecause it is easily quantified by quantification of MDA derivativeswith TBA (Thiobacbituric Acid), and this quantification ofderivatives can use a very simple, low cost, convenient spectroscopicmethod, 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, hasalso been used in many studies to evaluate on colon cancer patients.MDA was quantified in serum, plasma, red blood cells, urine and colontissue to make comparisons between the two groups: 1 group is coloncancer patients, 1 reference group is healthy persons, as well asexplore the relationship between MDA index and pathological factors
in colon cancer
The changes in oxidative stress have been studied after surgery forlung cancer, breast cancer and also studied after surgery for colon cancer.All studies have shown that oxidative stress occurs right after surgery forcolon cancer and tends to decrease over time after surgery, however, there
is a difference among studies in time and evaluation index
Trang 10Studies 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 surgicaloutcomes has not been mentioned in any study
In Vietnam, there have been no studies finding out about MDAcontent 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 AbdominalSurgery - 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 InternationalClassification of Diseases for Oncology, 3rd Edition of the WorldHealth Organization, the position of the tumor in the colon cancerdetermined from cecum to the end of the sigma colon, above therectosigmoid junction In this study, the tumor was identified as of thecolon, with the tumor position 15cm away from the anal margin
- Colon cancer patients at stages I, II and III The anatomicalresult on the tumor cell morphology is adenocarcinoma
- Carry out radical surgery to remove the tumor in the colon, with asurgery minutes describing in details the lesions and techniques
Trang 11- Follow the procedure of treatment, nursing before, during andafter surgery, have complete medical records and voluntarily agree togive disease samples for research
2.1.2 Exclusion criteria
- Recurrent colon cancer or secondary colon due to that cancerfrom
other organs metastasize
- Colon cancer has been treated with chemicals and radiationtherapy before surgery
- Colon cancer must undergo emergency surgery
- Colon cancer associated with other diseases combined (diabetes,cardiovascular disease, systemic disease …), smoking history, BMI >30
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
Trang 12content 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 reactiontakes 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, thecorresponding MDA amount in the reaction solution will bedetermined, 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 indexdetermination are provided by the Department of AnatomicPathology, Military Hospital 103 The colon segment containing thetumor after being removed from the patient via surgery will be coldstored in an ice box (without formalin) and taken to the Department
of Anatomic Pathology Here, tissue samples are taken at twolocations: diseased tissue is taken at the tumor and normal tissue istaken at a location at least 5cm from the tumor edge The tissuesamples were then stored at minus 1960C with liquid nitrogen beforebeing taken to oxidative stress testing The method of quantifyingMDA 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
Trang 13Blood samples of patients with colon cancer for MDA indexdetermination are taken from peripheral veins Next, the bloodsample was centrifuged at a speed of 3000 rpm for about 10 minutes
to separate plasma and red blood cells The red blood cells werestored at 40C before being analyzed for MDA index Thedetermination 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 todetermine the MDA content on erythrocyte membrane, it is necessary
to determine the protein content of the erythrocyte membrane andMDA 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
3.2.5.2 Criterion on early results after surgery
Including criteria: complications in surgery, death - complicationsafter surgery, time of postoperative ileus, number of fever days aftersurgery, 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 beforesurgery taken at the time before surgery The MDA concentration in thetissue was measured in µg/g sample, the MDA concentration of peripheralred blood cells before surgery was measured in µg/mg Protein
2.2.5.4 Criterion on changes of erythrocyte MDA content after surgery
Trang 14Peripheral erythrocyte MDA at 3 times: 1 day after surgery (after
24 hours), 3 days after surgery (after 72 hours) and 7 days aftersurgery (after 168 hours) The MDA content of blood samples aftersurgery 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 researchdoes 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: normal62.2%; 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%) CEAconcentration 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: 43patients (58.1%), sigma colon cancer accounts for the highest rate: