الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
Cancer has a complicated etiology, and the risk factors for cancer are ingrained in genetics and environment. Globally, an estimated 19.3 million new cancer cases and approximately 10 million cancer deaths occurred in 2020. Female breast cancer has surpassed lung cancer as the most commonly diagnosed cancer (11.7%), followed by lung (11.4%) and colorectal cancer (10.0%) in the third place. Colorectal cancer (CRC) is third in terms of recognition (6.1%) and second in terms of mortality (9.2%). Disease progression in cancer patients is determined not only by the histologic and molecular features of the tumor but also by the host response. Histologic distributions of tumor infiltrating lymphocytes (TIL) in microenvironment can be correlated with clinical stage and outcome in colorectal cancer patients. The presence of CD8+ T lymphocytes has been associated with good prognosis in different types of solid tumors. The association between tumor cell expression of FoxP3 and the tumor infiltration by FoxP3 expressing T lymphocytes with prognosis still controversial.
Colorectal Cancer (CRC) is associated with the population dietary and lifestyle factors. The association between colorectal cancer mortality and modifiable lifestyle factors is growing in evidence. Changes in individual health behaviors both before and after a diagnosis of colorectal cancer may improve outcomes of survivors tumor-infiltrating lymphocytes (TILs) have been reported to have a crucial effect on tumor progression and the clinical outcome in various types of cancer, including non-small cell lung cancer (NSCLC), colorectal, esophageal, and urothelial cancers and melanoma. Cancer cells are known to express tumor-associated antigens (TAAs) and chemokines, which become targets of the T cell-mediated immune response. T cells can be subdivided into functional subtypes, including cytotoxic CD8+ T cells (CTLs),) and regulatory T cells (Tregs; forkhead box P3 [FOXP3]).
Regarding CRC, CD8+ T cells are essential in recognizing and lysing the malignant cells in microenvironment. FOXP3+ T cells inhibit T-helper1 (Th1) and T-helper2 (Th2) as well as T-helper17 (Th17) in CRC.
The present case control study included 60 CRC patients and 60 control subjects matched by age and sex. Cases were sought from private laboratories between 2018 and 2022.
The tool used for collection data about patients’ life style was a telephonic Questionnaire. Patients contact information were detected from their clinical files, 100 patients were recruited through convenience sampling technique which is a non-probability sample characterized by being ease of access Finale number of patients were 60, questionnaire was done through telephone interview after taking written consent from patients. We retrospectively obtained tumor paraffin blocks and reviewed the database of these chosen 60 cases that underwent curative surgery for CRC from private laboratories. The personal data of the patients were replaced by numerical codes for privacy and confidentiality. This study was approved by the ethics committee of National Research Centre (Approval No. 19/027)
Inclusion criteria
• Patients that underwent radical surgical resection of CRC. with no other cancers or diseases, such as acute infection or diabetes, and they had not received previous radiotherapy or chemotherapy at the time of the surgery.
Exclusion criteria
patients received neoadjuvant radiotherapy because of the possible interference between this therapy and the assessment of the local immune response, and they Patients had other cancers or diseases, such as acute infection or diabetes.
Questionnaire included
Socio-demographic characteristics age, gender, residence which were divided to urban and rural and occupation).
Clinical data concerning disease (its duration and CRC site)
Life style factors concerning dietary history – dietary pattern during 2 years prior to cancer diagnosis was assessed through the dietary questionnaire introduced by Melbourne University (Giles GG, 2002). This food frequency questionnaire includes: Dairy products, as cheese, milk and yoghurt. Processed and red meat, Canned and fresh fish, as well as fruits, vegetables, and, drinks, also the questions were about fast food, food rich in calcium, spicy food and preserved food. The analysis of each food item includes the frequency of intake daily, weekly or monthly; the number of servings for each food item and conversions of household measures and serving sizes of various foods into grams. Life style factors concerning smoking history. Asking about history of physical activity done after explaining WHO definition of it which is. Anthropometric measurements included weight and height to calculate BMI.
PathologicalData
Gross description of the tumor; tumor size, site, number of metastatic lymph nodes, and with or without distant metastases.
Histopathological Examination
Three sections (4-5 µm thick) were cut from each block. One section was stained with hematoxylin and eosin for histopathologic evaluation. The other two sections were mounted on positively charged glass slide for immunohistochemical staining. The histopathological characteristics were reviewed. The histological diagnosis was established and verified by two pathologists. Clinicopathologic data were retrospectively collected from medical records and are summarized.
immunohistochemical staining
Conducting immunohistochemical staining on paraffin embedded sections by anti-FOXP3, and CD8 antibodies. The slides were incubated at 37°C overnight for accurate adhesion of the section of the slide. For CD8 staining Deparaffinization, rehydration, and epitope retrieval were performed in Pt link retrieval system (Dako, Copenhagen, Denmark). The staining steps and incubation times were preprogrammed into the auto Stainer Link software (Dakoomnis, Copenhagen, Denmark). Diluted primary antibodies against CD8 (Thermo Fisher Scientific, Runcorn, Cheshire, UK) and horseradish peroxidase labeled secondary antibody (Thermo Fisher Scientific) were used. For negative control, the primary antibody was replaced by normal mouse serum. Diaminobenzidine was used for color development and hematoxylin as counterstain. For Foxp3 staining tissue sections were dewaxed, and the antigens were exposed by citrate treatment (pH 6.0). Sections were then incubated in 0.3% H2O2 at room temperature and washed with distilled water and 0.1 M phosphate-buffered saline (PBS) before an overnight incubation in 10% goat serum and primary antibody (1:200, mouse anti-human FOXP3 monoclonal, produced by Abcam, purchased from Wuhan Amyjet Scientific) at 4°C. Subsequently, sections were treated with the secondary antibody (1:300, Goat Anti-mouse IgG Polyclonal Antibody). Staining color was developed with DAB substrate [diaminobenzidine (DAB) color kit (Beijing Zhongshan Golden Bridge Biotechnology). Tissues were counterstained with hematoxylin. PBS was used as negative control for the primary antibody.
o Image analysis immunoscoring
Immunostaining was visualized and photographed under a light microscope Olympus CX-41 with DP 12 Olympus digital camer(Olympus Optical Co. Ltd, Tokyo, Japan). Quantitative evaluation was performed by screening the sections and selecting at least five different fields with the high density of TILs. The density of CD8+ TIL and the positive expression of FoxP3 in tumor cells and tumor infiltrating lymphocytes were determined using the Leica Q win 500 image analysis system (LEICA Imaging Systems Ltd, Cambridge, England) which consists of Leica DM-LB microscope with JVC color video camera attached to a computer system. We selected five tumor fields with the highest expression and assessed the area percentage of the positive cells at high magnification (×200). We detected the positively stained cells, which were masked automatically by a blue mask called binary image. We used the measure field software program that automatically measures the area percentage of the detected features in the binary image and then displays the results in a table form. The immune score for FOXP3 positive cells and CD8+Tcells was evaluated according to (Cho et al.,2003) into scanty, moderate, and abundant expression.
Statistical analysis
was performed using SPSS 13 and quantitative data (mean and standard deviation), qualitative data (frequency distribution) were measured. Chi square test and Fisher’s exact test was used to compare proportions.
Chi-square (x2) test of significance was used in order to compare proportions between qualitative parameters.
The confidence interval was set to 95% and the margin of error accepted was set to 5%. So, the p-value was considered significant as the following:
Probability (P-value)
– P-value <0.05 was considered significant.
– P-value <0.001 was considered as highly significant.
– P-value >0. was considered 05 insignificant.
Results
The most significant dietary and life style CRC risk factors were higher consumption of red meat, preserved food (Daily consumption 36.8% of patients vs 1.67% of control), Daily spicy food consumption (60%of patients vs 5% of control), About most (80%) of patients consumed soft drinks daily compared to 45% of controls. 40% of CRC patients were smokers in comparison to (18.33) % of healthy control. The most significant protective factors were physical activity, higher consumption of fruits and vegetable as 60% of controls ate more than five servings of fruits and vegetables per day compared to 10% of patients, fresh fish and fruit juice (Daily consumption of fruit juice was higher among controls (40%) than among CRC patients (5%) and all these differences were represented as statistically significant p value = (p < 0.001).
CD8+ TIL in present study was insignificantly correlated with the clinicopathological parameters. FOXP3 expression was significantly correlated with tumor grade, nodal status, distant metastasis, tumor stage and DUKes’ classification. These data suggest that the presence of FoxP3 high expression is associated with good prognostic indicators.
Conclusion
This study provides supporting evidence that lifestyle and dietary modification are important factors in the prevention of colorectal cancer.
The presence FoxP3 expression in CRC is correlated with the favorable pathological prognostic parameters. Cancer colon progression is influenced by host immune response. Further studies are recommended to assess the role of tumor microenvironment in CRC prognosis.
Recommendations
2- People have to improve their physical activity, eat more fruits and vegetable, fresh fish and fruit juice.
3- Stop smoking most important life style must be changed to reduce CRC colorectal cancer .
4- Reducing consumption of red meat, preserved food,& daily spicy food will decrease CRC incidence in Egyptian population .
5- Therapytargeting FOXP3 will be helpful in treatmentstrategies.