|Year : 2019 | Volume
| Issue : 2 | Page : 194-200
DNA repair capacity of the colorectal cancer patients and the correlation between the pathological parameters
HK Tolan1, A Tozan-Beceren2, S Sardas2, O Senkesen3, C Celikel4, R Gencosmanoglu5, C Yegen6
1 Department of General Surgery, Health Sciences University Umraniye Training and Research Hospital, Umraniye, Istanbul, Turkey
2 Department of Toxicology, Marmara University-Faculty of Pharmacy, Istanbul, Turkey
3 Radiation Oncology Department, Acıbadem Altunizade Hospital, Istanbul, Turkey
4 Pathology Department, Marmara University Medical Faculty, Istanbul, Turkey
5 General Surgery Department, Acibadem Altunizade Hospital, Istanbul, Turkey
6 General Surgery Department, Marmara University Medical Faculty, Istanbul, Turkey
|Date of Acceptance||27-Aug-2018|
|Date of Web Publication||7-Feb-2019|
Dr. H K Tolan
Department of General Surgery, Health Sciences University Umraniye Training and Research Hospital, Umraniye, Istanbul 34764
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Colorectal cancers are third most common cancer in both genders. They are associated with genetic and environmental factors. Staging is important in the prognosis. Carcinoembryonic Antigen (CEA) provides preliminary information and there is a correlation between Proliferation Index (PI) and prognostic variables. Our aim is to investigate the relationship between DNA repair capacity and clinico-pathologic factors. Patients and Methods: The blood samples taken from cancer patients were irradiated. DNA repair capacity by comet technique was calculated. The CEA values were recorded. Pathology reports were collected and PI values were calculated. sResults: Total of 30 patients; male (n: 14) and female (n: 16) with a median age of 66.37 ± 10.32 were included. Mean CEA value was 42.85 (1.46 - 422.30 μgr/ml) μgr/ml. Mean % DNA repair capacity was 44.49 ± 5.24. In the pathology; 21 (70%) were T3 tumors; 18 (60%) had lymphatic and 12 (40%) had vascular 2 invasion. Perineural invasion was present in 8 (26.7%). According to the proliferation index (PI); 16 (53.3%) were in high percentile (PI > 66%) group. Conclusions: There was a significant correlation between; perineural invasion and tumor grade (P = 0.043); lymphatic and perineural invasion (P = 0.006); lymphatic invasion and vascular invasion (P = 0.034) and the DNA repair capacity with the lymphatic invasion (P = 0.026). There was also a statistically significant (P = 0.044) relationship between PI and lymphatic invasion. As a result in colorectal cancer patients DNA repair capacity can be used as a biomarker in the staging and also in the prediction of the tumor behavior.
Keywords: Colorectal cancer, DNA repair, proliferation index, Ki67
|How to cite this article:|
Tolan H K, Tozan-Beceren A, Sardas S, Senkesen O, Celikel C, Gencosmanoglu R, Yegen C. DNA repair capacity of the colorectal cancer patients and the correlation between the pathological parameters. Niger J Clin Pract 2019;22:194-200
|How to cite this URL:|
Tolan H K, Tozan-Beceren A, Sardas S, Senkesen O, Celikel C, Gencosmanoglu R, Yegen C. DNA repair capacity of the colorectal cancer patients and the correlation between the pathological parameters. Niger J Clin Pract [serial online] 2019 [cited 2019 Feb 17];22:194-200. Available from: http://www.njcponline.com/text.asp?2019/22/2/194/251780
| Background|| |
Colorectal cancer ranks third after the prostate and lung cancer making 8% of all new cancer cases in males, and 8% of all new cancer cases after breast and lung cancer in females. When we look at the data given in our country, colorectal cancers are the 7th among the most common seen 10 cancers and this disease causes about 3200 deaths per year. As it is in the whole world, it is also an important health problem in our country in terms of mortality and morbidity.
Global increase in the knowledge about the molecular and biological properties of colorectal cancer has shed light on its pathogenesis. It has been reported that these cancers are the result of an interaction between both the genetic susceptibility and the environmental effects , The mucosa which is normal at the beginning is transformed into an adenoma by various factors; later this adenoma formed is transformed into a cancer over time as a result of some other ongoing mutations. In an epidemiological study conducted, it was reported that the incidence among genders was equal, the risk of colorectal cancer was increasing after the age of 40, and that the risk reached the highest level at 50-55 years of age. Research done suggests that the colorectal cancers are a result of both the hereditary sensitivities and the environmental factors. In a genetic model of a colorectal cancer, some key mutations have been shown to be leading to a colorectal cancer. A number of studies have been performed on genetic factors that cause this cancer and it has been reported that 25% of colorectal cancers are caused by hereditary factors. Another study that shows that the hereditary factors are important in colorectal cancer predisposition is the work of Foulkes and friends. They showed that the risk of developing cancer in the first-degree close relatives of the colorectal cancer patients is higher than the normal population. The molecular model of genetic changes in the colorectal adenoma-carcinoma chain was first described and defined by Vogelstein et al.
Staging in cases with colorectal cancer is of great importance in determining the prognosis of the patient and in deciding the need for an adjuvant treatment in these patients. Pathological staging is performed after the surgical exploration and the evaluation of the specimen after the surgical resection. The final staging of the cancer is based on the TNM system. It is also known that carcinoembryonic antigen (CEA), which can be tested before the surgery in the blood can provide a preliminary information about the resectability and the progression of the disease.
The purpose of this study is to evaluate the possible prognostic value of colorectal cancers' pathology reports and the blood CEA levels; comparing them with the bio-indicators used in the literature to investigate whether these bio-indicators can used in the colorectal cancer staging.
| Materials and Methods|| |
In this prospectively conducted study the DNA damage and DNA repair capacities of patients diagnosed with colorectal cancer who were referred to-General Surgery department were calculated. The pathology reports, blood test results and the demographical data of the patients were collected. Relationship between these findings and the aggressiveness of the tumor and the disease were evaluated.
Patients who meet the inclusion criteria of the study were included after taking the informed consent forms which they read and then signed for understanding the contents and principles of the study. Ethical approval by protocol no MAR-YÇ-2007-0281 has been obtained for the study. Thirty patients between January 2008 and August 2009 meeting the criteria were enrolled into the study.
- Patients diagnosed with a colorectal cancer
- Patients who signed the consent form
- Patient age 18 or more.
- Patients with familial colorectal cancer history
- Inflammatory bowel disease patients who developed colorectal cancer
- Patients who have received preoperative chemotherapy and/or radiotherapy
- Families with familial disease with known genetic transmission
- Patients who wish to quit at any stage
- Patients whose pathologic examination is to be performed outside our hospital.
Calculation of DNA damage and the DNA repair capacity
Calculations were made using the Alkali Comet Technique. Preoperatively, 5 ml of blood was taken into sterile heparinized tubes with a 21 G sterile injector, which was then sent to the Radiation Oncology Department, where they were radiated with 0.75 Gy; placed in a polyethylene water bath at 37 degrees centigrade to mimic body temperature for 28 seconds, which was calculated by a previously performed dose-viability study. Irradiated blood was taken to-of Pharmacy Laboratory of Pharmaceutical Toxicology Department in two separate eppendorfs. One of the eppendorf was incubated at 37 ° C for 30 minutes to allow the DNA to repair itself, and the other eppendorf was evaluated immediately with the comet technique without being allowed to repair the DNA damage. By this way the comet values in induced peripheral blood lymphocytes, comet values in induced blood, and the comet values in induced lymphocytes and waited for the repair were separately calculated.
Alkali comet technique application
Insolation of lymphocytes
Hundred microliters of blood from the laboratory (plain comet or induced) are mixed with 1 ml PBS (Phosphate Buffer Saline) as a buffering solution and then left to stand for 10 minutes on ice. Then 100 microliters of Histopaque (Lymphoprep) is added to each eppendorf precisely. Lymphoprep is necessary for layering of the lymphocytes. These eppendorfs are then centrifuged at 4°C for 5 min at 400 G to separate the lymphocytes in the blood.
When the eppendorfs removed after the centrifugation and are visually observed, the erythrocytes are seen as a red layer at the bottom. At the top, there is a cloudy area in which there is the serum seen as a yellow layer and lymphocytes in between these two layers. With the automatic pipette, the lymphocyte layer to be used in the comet is withdrawn and placed in a separate new eppendorf.
Following dissociation of lymphocytes, LMA (Low Melting degree Agar) (0.65 mg LMA dissolved in microwave in beaker with 100 ml PBS and stored at 40 degrees) is prepared to maintain the life of the lymphocytes. The temperature of this agar is adjusted to + 37 degrees and 100 microliters of agar per eppendorf is added for 100 microlitres of lymphocytes separated. This mixture is spread by a micropipette over the slides longitudinally, covered with coverslip and kept in a dark box at + 4 ° C for about 30 minutes until the agar frost to prevent DNA damage.
The slides were taken out and placed on the lysis solution (made by adding 60 ml of shakers, 53.4 ml of stock lysis solution + 6 ml of DMSO + 0.6 ml of Triton-X) prepared in the flasks and incubated overnight at + 4 ° C in the dark refrigerator.
The slides that are kept at + 4 ° C after the separation of the lymphocytes are removed from the lysis solution by the next morning and filled with electrophoresis solution (10 M NaOH 37.5 ml + 0.2 M EDTA and 1250 ml supplemented with distilled water). In order to separate the DNA strands, the chalks are left in the refrigerator for 20 minutes in the dark. Surrounded with crushed ice and filled with electrophoresis solution, the slides are carefully placed into the electrophoresis tank. The electrophoresis tank is placed in a dark room at 300 milliamp for 20 minutes and the DNA fragments are left to be electrophoresed [Figure 1].
The broken DNA fragments of the broken DNA strand are always negatively charged. As negatives in electrophoresis migrate towards the positive column, DNA fragments that drift on the lamina form the comet tail on the slides.
At the end of the procedure, the slides are made ready for the staining phase by washing the slides in the neutralization buffer (taking 48.5 grams of 0.4 M Tris den and diluting them to 1000 ml with distilled water) (pH = 7.5) for 3 minutes at intervals of 5 minutes + 4 degrees.
After the neutralization process, 50 microliters of Etidium Bromide (5 mg of Ethidium Bromide completing with 50 ml of distilled water), which is used as a fluorescent dye, is placed in the dark room on the slides with micropipelines and the surfaces are covered with the laminates. Etidium Bromide is a toxic and carcinogenic material at a later stage.
The process is done in a dark room in a completely isolated environment. These slides are stored in a closed box at + 4 ° C for 10 minutes. Later, the dark room is examined with a fluorescence microscope.
In this study, the scattered nuclei are selected from each of the 50 tubes of each patient in each study, and the data are recorded on the computer to be able to calculate later [Figure 2].
|Figure 2: Example view of comet and scattering under fluorescence microscopy|
Click here to view
After this process, measured values and necessary parameters are calculated by using BAB Bs Comet Assay software on the computer. In this way, comet in the normal blood of the patient with colorectal cancer, comet in the irradiated blood, and comet in the blood irradiated and then left alone rested for repair (Challenge) are calculated [Figure 3].
|Figure 3: The use of the program to calculate the values of selected cometes in a fluorescent microscope (×40)|
Click here to view
With all these results the % DNA repair capacities were calculated by the formula;
(%DNA irradiated comet - %DNA repaired comet) ×100/ %DNA irradiated.
Specimens of all patients were evaluated by-Faculty of Medicine Pathology department. The final pathology reports approved by our pathology were followed up and collected from our Pathology Department.
All the paraffin blocks of the patients who had been taken into the study were taken out from the Pathology Department archive and were re-examined by the same pathologist to select the sections suitable for Ki-67 immunohistochemical staining to calculate the proliferation index (PI). The most appropriate blocks were selected, and stained for immunohistochemistry as detailed below. All the patient samples were collected completely.
In formalin fixed tissues streptavidin biotin peroxidase immunohistochemical staining was applied to demonstrate Ki-67 immunosuppression. In this method, 3 μm thick sections were taken from the paraffin-embedded tissues, and 1 minute overnight at 37°C, followed by deparaffinization with three separate xylenes for five minutes. Sections were processed in two separate 96% ethanol solutions and the endogenous peroxidase activity in the tissue was suppressed by 3% H2O2 (in methanol). Sections were washed with distilled water were subjected to antigen retrieval with a pH 6 citrate buffer solution in a microwave to remove masked antigens.
The slides cooled in the room temperature for 20 minutes were washed with two separate phosphate buffer solutions (PBS) and the tissues were blocked with protein for 10 minutes to prevent non-specific staining.
After protein blocking, the sections were incubated in room temperature for 60 minutes with 1: 200 dilution of anti-Ki-67 (Clone SP6, Thermo Fisher Scientific, Fremont, CA, USA). At the end of the period, the sections were washed with two separate PBS and biotinylated secondary antibody (SensiTek anti-polyvalent HRP kit, ScyTek Laboratories, Utah, USA) was left for 10 minutes. After repeated washing with PBS, streptavidine peroxidase (SensiTek anti-polyvalent HRP kit, ScyTek Laboratories, Utah, USA) was incubated for 10 minutes. Diaminobenzidine (DAB) chromogen was added to make the dye visible. The distilled water-washed sections were stained with nuclear counterstaining with Mayer Hematoxylin and dehydrated by passing through ethanol. The xylanene was removed and the tissue covered with appropriate closure material was evaluated with light microscopy.
All preparations were evaluated semi quantitatively with the same pathologist at-Pathology Department. In total, an average of 2000 cells from each slide were counted from different areas and indexes were formed and these values were divided into three separate percentile groups to participate in the evaluation [Figure 4].
All of the results are expressed as mean ± standard deviation. A Pearson correlation test was used to compare the variables. Student's t test was used to compare clinicopathologic variables. All statistical analyzes were compared using SPSS 13.0 (SPSS Inc., Chicago, USA).
| Results|| |
In accordance with pre-determined criteria a total number of 30 patients; 14 male (46.7%) and 16 female patients (53.3%) were included in the study. The mean age of the patients was 66.37 (±10.32).
The preoperative mean CEA of the patients was found to be 42.85 μgr/ml (1.46 μgr/ml-422.30 μgr/ml). CEA hormone blood tests were all performed in our hospital lab with standardized instruments and kits calibrated routinely (normal values of CEA 0.0 to 3.4 μg/ml).
Similarly the results of the DNA repair capacities of the patients detected in the blood samples of patients prior to surgery and the mean DNA repair capacity percent of patients was found to be 44.49 ± 5.24% [Table 1].
When the final pathology reports of the patients were examined postoperatively, 21 (70%) of the patients who were taken into the study were found to have T3 tumors according to the TNM classification. When we look at the positive Lymph node status, 12 of the patients were N0 (40%) and other 12 were N1 (40%); the remaining 6 patients were N2 (20%) according to the TNM staging.
When pathological grades were evaluated; 19 (63.3%) of these patients were reported as having grade 2 tumors. This was followed by 10 grade 3 (33.3%) patients. One of the patients' grade was not mentioned [Table 1].
Staging of patients was performed in our hospital by looking at the American Joint Committee on Cancer staging criteria. When we looked at the stages of the patients who were followed up in our study; 22 of them were stage III (73.3%), 7 were diagnosed as stage II (23.3%) and 1 as stage I (3.3%) [Table 1].
Patients were also evaluated for lympho-vascular and perineural involvement, which is known to be important in the pathologic evaluation of the disease; the lymphatic invasion was seen in 18 patients (60%); and vascular invasion was seen in 12 (40%) patients. Perineural invasion was detected in 8 patients (26.7%) [Table 1].
When the proliferation index (PI) of Ki-67 were examined in the tumors of patients; 16 of the tumors (53.3%) were in PI >66% (Group 3) and 9 of them were in the PI group with PI <33% (Group 1) [Table 1].
| Discussion|| |
Many parameters can be studied both preoperatively and postoperatively and they may provide information about the prognosis and the life expectancies of the colorectal cancer patients. Particularly giving a neo-adjuvant treatment in advanced cases is important in the treatment of some subgroups of these cancers.
When we look at the distribution according to the gender of the patients participating in the study, we found that 14 (46.7%) were male and 16 (53.3%) were female. The mean age was 66.37 (±10.32) years. It is known that in the patients with colorectal cancer the preoperative carcinoembryonic antigen (CEA) level has a predictive value in the disease resect ability and tumor stage. However, in our prospective conducted study, there was no significant difference between the TNM stage, tumor grade and other clinicopathologic variables of the cancer and the CEA levels (P = 0.60). In our opinion this was due to the small number of patients in our study group (n: 30); and also because of the lack of the homogeneous distribution of the CEA levels of our patients.
Histologically grade of the adenocarcinomas are based on the presence of their glandular structures seen under the microscope. Grade has also a prognostic value in colorectal adenocarcinoma patients.,,, In our study, a statistically significant relationship was also found between the grade and the status of the tumor (P = 0.010) supporting the literature.
Perineural invasion in histopathological evaluation is another important criterion for pathological staging. According to literature, it has been shown that as the stage increases, the rate of perineural invasion increases and the regional recurrence of cancer increases which is a bad prognostic sign of the disease , In our study group also there was a statistically significant (P = 0.043) correlation between the perineural invasion and the grade of the tumor. Also between the lymphatic invasion and the perineural invasion (P = 0.006); and between the lymphatic invasion and the vascular invasion (P = 0.034) there was a statistically significant correlation. The TNM classification is the preferred staging system in colorectal cancers. In this system, the tumors penetration depth (T), the presence and absence of mesenteric nodes (N), and the presence of distant metastases (M) is evaluated. Pathological staging is performed according to the TNM system after the surgical extraction of the tumor and by the specimen assessment.
In the AJCC staging system, the stages were divided into subgroups considering the wall penetration and the lymph node involvement of the cancer. Survival also varies among these subgroups. The grade of lymphatic invasion and the presence of vascular invasion are important additions to the TNM staging system , We have achieved similar results with our work.
Proliferation index (PI) is used as a marker to assess how aggressive the tumor is and how it is susceptible to spread. The evaluation of the amount of proliferation of tumor cells pathologically is also an indicative of the aggressive behavior pattern of the tumor , In most retrospective designed studies, it was demonstrated that the amount of the proliferation could help us predicting the behavior of the tumor ,, There is a significant correlation between high PI values and poor prognostic variables in colorectal cancer. Various retrospective studies have demonstrated that high PI values are associated with invasion depth, lymph node metastasis, and distant metastasis of various tumors. Colorectal cancers have been shown to be involved in pathologic characterization of tumor differentiation with PI as a metastatic disease and/or locally invasive. Several studies have shown in the literature that there is a significant relationship between penetration depth of the tumor, frequency of lymph node metastasis and frequency of distant metastasis as the PI value increases ,,, Also some published studies also have shown that there is a relationship between the PI values and the prognosis of the colorectal cancers ,, In our study, similar to the literature, a statistically significant relationship (P = 0.015) was found between the disease and the TNM stage of the cancer. As an additional data to this literature, there was a statistically significant (P = 0.044) relationship between PI value and histopathological lymphatic invasion of the tumor in our study. When all these findings are combined all together, it can be seen that there is a significant relationship between the high PI values of colorectal cancers and the poor pathologic findings.,,,
The DNA damage and repair (Comet Assay) tests have been used in the literature to assess the geno-toxicity. Also the Challenge technique is used extensively as a biomarker with mutagenicity tests.,, In our study, we looked at the DNA repair capacities of the colorectal cancer cases and try to see whether there is a predictable relationship between this amount of repair capability and their clinicopathologic factors. When compared with all the other parameters, it was found that there was a statistically significant (P = 0.026) relationship between the DNA repair capacity of the patients and the lymphatic invasion of the cancer. Especially we think that if sufficient number of subgroups could be studied the DNA repair capacities that could be tested pre-operatively and non-invasively; this may be useful for the prediction of the advanced groups in which the neo-adjuvant treatment is important before the surgery in colorectal cancer patients. The limitation of our study is that; to be able to use this DNA repair capacity technique there is need for multiple laboratory facilities and special devices as detailed above, thus lack of these may make it impracticable to be performed in a routine hospital setting.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]