GENETIC POLYMORPHISM OF SEVERAL INTERLEUKINES INVOLVED IN THE RELATION INFLAMMATION COLO-RECTAL CANCER

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1 UNIVERSITY OF MEDICINE AND PHARMACY FROM CRAIOVA THEODOR DUMITRESCU, PhD student GENETIC POLYMORPHISM OF SEVERAL INTERLEUKINES INVOLVED IN THE RELATION INFLAMMATION COLOREAL CANCER ABSTRA Scientific Coordinator Prof. Univ. Dr. ION GEORGESCU CRAIOVA 20

2 CONTENTS INTRODUION 3 I. STATE OF KNOWLEDGE CHAPTER 1. EPIDEMIOLOGY AND ETIOLOGY FOR COLOREAL CANCER CHAPTER 2. ANATOMOPATHOLOGY OF COLO REAL CANCER CHAPTER 3. THE INVOLVEMENT OF CYTOKINES IN NEOPLASMS II. SPECIFIC CONTRIBUTIONS CHAPTER. COLOREAL CANCER, A CLINICAL AND STATISTIC STUDY CHAPTER 5. INVESTIGATION OF THE CORRELATION BETWEEN EIGHT POLYMORPHIC VARIANTS OF CYTOKINES IL1B, IL1RN, ILR, IL8, IL10 ŞI TNF 6 ALFA AND COLOREAL CANCER CHAPTER 6. CONCLUSIONS 12 SELEIVE REFERENCES 13 2

3 INTRODUION Colorectal cancer (CRC) is a major health burden worldwide, being the most common form of cancer and the second leading cause of cancerrelated deaths in Europe, with the highest mortality rates in both genders in Central and Eastern Europe. Although mortality has been declining over the last two decades, prognosis strongly depends on the stage at diagnosis, patients diagnosed at an early stage of the disease have a favorable prognosis. The vast majority of CRC cases arise sporadically, although inherited susceptibility have been estimated to account for 35% of the risk of CRC. These inherited syndromes (familial adenomatous polyposis and the hereditary nonpolyposis CRC) are responsible for about 5% of CRC. Classically, CRC results from accumulation of genetic and epigenetic events that modulates the process of transformation of normal glandular epithelial cells to adenocarcinomas. The inflammatory conditions like Crohn disease and ulcerative colitis increase the risk of CRC. A lower risk of CRC was observed for regular users of antiinflammatory drugs. The connection between inflammation and cancer is generally accepted and accumulating evidences shows that cancer arises and progresses in chronically inflamed tissue, and this is particularly notable in the gastrointestinal tract. Interindividual variability on intensity and magnitude of inflammatory response could be influenced by single nucleotide polymorphisms located in cytokines genes and their receptors, especially in promoter regions. The genetic variations determine changes in the tumor microenvironment and result in increased susceptibility and/or poor prognosis in gastrointestinal cancers, including CRC. Development of molecular technology has led to a better understanding of the mechanisms of tumor development and progression. But current information is incomplete and sometimes divergent. Multiple molecules such as oncogenes, growth factors, tumor suppressor genes, cytokines and other genes and regulating proteins are involved in development of colorectal cancer. In this context, this paper aims to present initially a clinical study based on statistical parameters of colorectal cancer. Then, we analyse in detail the correlation between eight polymorphic variants of six interleukins: IL1B, IL 1RN, ILR, IL8, IL10 şi TNFα (IL1B: 31T>C rs362; IL1B: 5C>T rs9; IL1B: 395C>T rs363; IL1RN: 2018T>C rs19598; ILR: 3223C>T rs20568; IL8: 251T>A rs03; IL10: 1082A>G rs ; TNFα: 308G>A rs ) and colorectal cancer. To assess the 8 polymorphisms we used realtime PCR technology with TaqMan probes (Applied Biosystem) specific for cytokines gene transcription. Validation of the gene expression patterns that we proposed, with important role in the initiation and development of colorectal malignant processes will contribute to future early diagnosis, to estimating efficiency of therapeutic measures and improving prognosis evaluation. Keywords: colorectal cancer, polymorphisms, cytokine, RealTime PCR. 3

4 I. STATE OF KNOWLEDGE CHAPTER 1. EPIDEMIOLOGY AND ETIOLOGY FOR COLOREAL CANCER presents data on the incidence of colorectal cancer in Romania and worldwide, pathology representing a major public health issue. (figure 1) Figure 1. Incidence and mortality of neoplasms worldwide, for both genders (after International Agency for Research on Cancer 2008) Here there are shown disease epidemiology, clinical risk factors, protective factors and colorectal cancer precursor lesions, emphasizing the key role of environmental factors. CHAPTER 2. ANATOMOPATHOLOGY OF COLOREAL CANCER is a summary of the classifications of colorectal cancer by cell type, layout structure, growth and invasion. It insists on the detailed TNM staging, after AJUI. CHAPTER 3. THE INVOLVEMENT OF CYTOKINES IN NEOPLASMS treats, on the one hand, the theory that inflammation represents a precursor for colorectal cancer and, secondly, shows the involvement of cytokines in cancer development, with their pathological implications and provides a detailed description of the six interleukins whose genetic polymorphism was investigated by us in relation to colorectal cancer. II. SPECIFIC CONTRIBUTIONS CHAPTER. COLOREAL CANCER, A CLINICAL AND STATISTIC STUDY Colorectal cancer represents nowadays the most frequent localization for digestive neoplasm, with an increased incidence worldwide. Though CRC is a tumor of extreme gravity, it can progress without symptoms frequently. Clinical signs suggesting the disease appear lately and the diagnosis is set rarely in the therapeutic state, most of colorectal cancers being advanced when diagnosed. This means the diagnose should be set before the symptoms appear using the screening concept. Material and methods The study was conducted on a group of 136 patients from II nd Surgical Clinic from Clinical Emergency Hospital of Craiova, all with diagnosis of colorectal neoplasm, hospitalised on a period of almost 3 years from 1 st of October 2008 until 31 th of August 20. All patients benefit of surgery, paleative or curative resections beeing possible for all cases. There have been performed right and left hemicolectomies, segmentary, subtotal and total colectomies, rectosigmoidian resections and amputations. We followed many parameters, among which: gender, age, localisation of the tumor, surgical procedure, anatomopathological diagnosis and ptnm staging, postoperative complications and presence or absence of invasion on the resection edges.

5 Results and discussions In most cases, the neoplasm was located distal, the most frequent situation being rectal, with 6 cases (33,82%), followed by the sigmoidian localization with 28 cases (20,58%) and rectosigmoidian jonction localization with 18 cases (13,23%). We notice that, in our study, the next localization by frequency is transvers colon (15 cases,03%). Table 1. General postoperative morbidity Number of Postoperative complications cases Supurative complications 18 13,23% 12,2% from all anastomosis Anastomotic leakage 12 Peritoneal abcess 3 2,2% Peritonitis 3 2,2% Urinary infection 15,03% Tromboembolic complications 5 3,6% Sepsis (MODS) 2 1,% Mortality 2 1,% The 3 postoperative complications (anastomotic leakage, peritoneal abcess and peritonitis) represent different evolutive possibilities of anastomotic breakdown, which is the most frequent cause of morbidity and mortality in colorectal surgery. (table 1). Conclusions Colorectal cancer occupies the first place among digestive neoplasms, with an increasing incidence in our country nowadays. The main treatment in CRC is represented by surgery, the response to oncological treatment being poor. Screening for colorectal cancer is another important matter, which may help discover the disease in an early stage when surgery can provide complet recovery. We emphasize the important role of anatomopathology department which is available to study the resection edges and the colorectal tumor. 5

6 CHAPTER 5. INVESTIGATION OF THE CORRELATION BETWEEN EIGHT POLYMORPHIC VARIANTS OF CYTOKINES IL1B, IL1RN, ILR, IL8, IL10 ŞI TNFALFA AND COLOREAL CANCER This chapter represents the basis of this thesis. Our study aims were to investigate the correlation between eight polymorphisms of six cytokines (IL1B, IL 1RN, ILR, IL8, IL10 and TNFα) and colorectal cancer, so that to determine the risk of developing this illness according to our studied SNPs in Romanian population. (table 2) GENE FUNION POLYMORPHISM SNP LOCALIZATION (database) IL1B Proinflammatory 31T>C rs362 promoter IL1B Proinflammatory 5C>T rs9 promoter IL1B Proinflammatory +395C>T rs363 IL Proinflammatory +2018T>C rs RN ILR Antiinflammatory 3223C>T rs20568 promoter IL8 Proinflammatory 251T>A rs03 promoter IL10 Antiinflammatory 1082A>G rs promoter TNFα Proinflammatory 308G>A rs promoter Table 2. Polymorphisms SNP of cytokines investigated in our study Material and methods We included in this hospitalbased casecontrol study a total of 3 Romanian subjects (1 patients diagnosed with sporadic CRC and 233 controls). (table 3) All CRC cases were established by standard diagnostic procedures (physical exam, digital rectal examination, fecal occult blood test, sigmoidoscopy, colonoscopy, barium enema, imaging tests) and confirmed histopathologically in the Clinical Hospital of Craiova, Romania. Age and gender matched controls of the same ethnic and geographical origins were recruited among unrelated volunteers admitted to the same hospital. Blood samples were obtained from both groups and demographic characteristics, age, sex and family history of cancer or other diseases were recorded for each subject. The subjects with a positive history of a tumor, autoimmune, inflammatory or infectious chronic disease were excluded. The Ethics Committee of University of Medicine and Pharmacy of Craiova, Romania approved this study and written informed consent was obtained from all the subjects. Table 3. Subjects characteristics Variable Colorectal cancer Control N Male/Female 8/5 13/90 Age (years), mean±sd 65,91±,1 63,69±,9 6

7 Location cecum sigmoid descending transverse ascending rectum Tumor stage Dukes stage A + B C + D Unknown/Not defined Differentiation Grade G1 well G2 moderate G3 poor Samples were collected in RNA Later Solution, Ambion and stored at C for 122 hours and then transferred to 80 o C. To isolate genomic DNA from blood and tissue samples collected we used the Wizard Genomic DNA purification kit (Promega, Madison, WI). To highlight a single nucleotide polymorphisms (SNP), we used TaqMan Universal Master Mix and TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA) specific for each of the polymorphisms studied. Statistical analysis Hardy Weinberg equilibrium was tested among controls using χ2 tests. Allele and genotype frequencies were compared between groups by chisquare test. Associations between genotypes and CRC were calculated as odds ratios (OR) with 95% confidence intervals (CIs) using unconditional logistic regression analysis, with gender and age adjustment. In addition to the overall association analysis, we performed a stratified analysis by tumor stage and histology to further explore the association between cytokine genotypes and the risk of CRC in each stratum. In all cases, homozygous genotype for the most common allele in Caucasians was used as the reference category (codominant and dominant models). A Pvalue less than 0.05 was considered statistically significant. All data analysis was performed using SPSS v.1.0 software package (SPSS Inc., Chicago, IL, USA). Results and discussions For each polymorphism of the eight studied we proceed following the same steps. First of all, we genotyped the subjects (colorectal cancer group and control group), then we analysed statistical associations between a certain genotype and risk of developing CRC.

8 Table. Risk of CRC by genotype Polymorphism IL1B 31 T>C C carriers Colorectal cancer (n=1) 6 (.%) 6 (.%) (.12%) 80 (55.56%) Control (n=233) OR (95%CI) p value 10 (5.92%) 105 (5.0%) 21 (9.01%) 126 (5.02%) 1.02 ( ) 1.2 ( ) 1.06 ( ) IL1B +395C>T T carriers 8 (60.2%) 6 (31.9%) (.6%) 5 (39.58%) 122 (52.36%) 93 (39.91%) 18 (.3%) 1 (.6%) 0.1 ( ) 0.86 ( ) 0.2 ( ) IL1B 5 C>T T carriers 65 (5.1%) 63 (3.5%) (.%) 69 (5.86%) 108 (6.35%) 99 (2.9%) 26 (.%) 125 (5.85%) 1.06 ( ) 1.02 ( ) 1.05 ( ) IL1RN +2018T>C C carriers 5 (52.08%) 5 (3.50%) 15 (10.2%) 69 (.92%) 133 (5.08%) 89 (38.20%) (.2%) 100 (2.92%) 1.08 ( ) 2.2 ( ) 1.22 ( ) ILR 3223C>T T carriers 9 (5.86%) 51 (35.2%) 1 (9.2%) 65 (5.1%) 13 (5.51%) 85 (36.8%) 1 (6.01%) 99 (2.9%) 1.02 ( ) 1.0 (0. 3.) 1. ( ) IL8 251T>A TA A carriers 50 (3.2%) 6 (6.53%) 2 (18.5%) 9 (65.28%) 81 (3.6%) 106 (5.9%) 6 (19.5%) 152 (65.2%) 1.02 ( ) 0.95 ( ) 1.01 ( ) IL A>G AG GG G carriers 65 (5.1%) 60 (1.6%) 19 (13.19%) 9 (5.86%) 80 (3.33%) 8 (50.6%) 35 (15.03%) 153 (65.6%) 0.63 ( ) 0.6 ( ) 0.6 ( ) TNFA 308 G>A GG GA A carriers 5 (9.86%) 26 (18.06%) 3 (2.08%) 29 (20.1%) 189 (81.12%) 2 (18.02%) 2 (0.86%) (18.88%) 1.02 ( ) 2. ( ) 1.08 ( ) Analysing the results (table ), we can see there is a statistical association with colorectal cancer only for two polymorphisms: IL1RN +2018T>C and IL A>G. A significant association has been observed for IL1RN +2018T>C polymorphism and CRC, the relative risk for carriers genotype was 2.2 (OR 2.2, 95% CI: ) when compared with the more frequent genotype. In a recessive model, the carriers of C allele were not associated with CRC (OR 1.22, 95% CI: ). Also, a significant association was observed for IL A>G polymorphism: the subjects carrying AG genotype were at a lower risk for CRC (OR 0.63, 95% CI: , p=0.01), when compared with the more frequent genotype. Furthermore, in a dominant model the carriers of G allele were protected against CRC (OR 0.6, 95% CI: ). Having these results, in addition to the overall association analysis, we performed a stratified analysis by tumor stage, histology and localization of the tumor to further explore the association between cytokine genotypes and the risk of CRC in each stratum. 8

9 Table 5. Comparative analysis between genotype frequencies and the risk of CRC in the tumor stage subgroups Polymorphism Tumor stage I+II n=5 (%) Tumor stage III+IV n=6 (%) IL1B 31 T>C C carriers 3 (9.33%) 31 (1.33%) (9.3%) 38 (50.6%) 0.85 ( ); ( ); ( ); (39.06%) 30 (6.88%) 9 (1.06%) 39 (60.9%) 1.22 ( ); (0.5.8); ( ); 0.33 IL1B +395C>T 1 (5.6%) 2 (36.00%) (9.33%) 0.86 ( ); ( ); (65.63%) 18 (28.13%) (6.25%) 0.56 ( ); ( ); 0.5 IL1B 5T>C 3 (9.33%) 31 (1.33%) (9.3%) 0.91 ( ); ( ); (0.63%) 29 (5.31%) 9 (1.06%) 1.22 ( ) ; ( ) ; 0.2 IL1RN +2018T>C 0 (53.33%) 26 (3.6%) 9 (12.00%) 0.9 ( ); ( ); (8.%) 2 (2.19%) 6 (9.38%) 1.30 ( ) ; ( ) ; 0. ILR 3223C>T T carriers 2 (56.00%) 26 (3.6%) (9.33%) 33 (.00%) 0.98 ( ); ( ); ( ) ; (53.12%) 23 (35.9%) (10.9%) 30 (6.88%) 1.0 ( ); ( ); ( ); 0.53 IL8 251T>A TA A carriers 30 (0.00%) 33 (.00%) 12 (.00%) 5 (60.00%) 0.8 (0. 1.9); ( ); (0. 1.3); (29.69%) 32 (50.00%) 13 (20.31%) 5 (0.31%) 1.29 ( ); ( ); ( ); 0.5 IL A>G AG GG G carriers 3 (5.33%) 30 (0.00%) (1.6%) 1 (5.6%) 0.60 ( ); ( ); ( ); (5.31%) 2 (2.19%) 8 (12.50%) 35 (5.69%) 0.63 ( ); ( ); ( ); 0. TNF A 308 G>A GG GA A carriers 65 (86.6%) 9 (12.00%) 1 (1.33%) 10 (13.33%) 0.62 ( ); (0.13.3); ( ); (1.88%) (25.00%) 2 (3.13%) 18 (28.13%) 1.5 ( ); ( ); ( ); 0. A positive association was found only for IL1RN genotype and that was limited to I and II stages (OR 2.2, 95% CI: ). (Table 5). The subclassification of CRC by TNM stage did not show any association with CRC for the other SNPs studied. 9

10 Table 6. Comparative analysis between genotype frequencies and the risk of CRC in the histologic grade subgroups Polymorphism G1 N= 3 G2 N= 80 G3 N= 2 IL1B 31 T>C C carriers ( ); ( ); ( ); ( ) ; ( ) ; ( ) ; ( ) ; ( ) ; ( ) ; 0.61 IL1B +395C>T ( ) ; ( ) ; ( ) ; ( ) ; ( ) ; ( ) ; 0.39 IL1B 5 C>T ( ); ( ); ( ) ; ( ) ; ( ) ; ( ) ; 0.96 IL1RN +2018T>C ( ) ; ( ) ; ( ); ( ); ( ) ; ( ) ; 0.1 ILR 3223C>T T carriers ( ) ; (0. 8.6) ; ( ) ; ( ) ; (0.63.0) ; ( ) ; ( ) ; ( ) ; ( ) ; 0.86 IL8 251T>A TA A carriers (0.62.2) ; ( ) ; ( ) ; ( ); ( ); ( ) ; ( ) ; ( ) ; (0.2.81) ; 0.19 IL A>G AG GG G carriers ( ); ( ); ( ) ; ( ); ( ); ( ) ; ( ) ; (0. 1.5) ; ( ) ; 0.0 TNF A 308 G>A GG GA A carriers ( ); ( ); ( ) ; ( ); ( ); ( ) ; ( ) ; 0.33 / 1.50 ( ) ; 0.38 In a stratified analyse according to tumoral grading, the only association between CRC and cytokine polymorphisms was found for carriers of IL G allele and was restricted to poorly differentiated cases G3 (OR 0.36, 95% CI: ) (Table 6). No significant differences were observed between histologic grading and controls for the other seven polymorphisms. 10

11 Table. Association between IL1B and IL1RN polymorphisms and colorectal cancer in the tumor site subgroups Polymorphis m IL1B +395C>T N = Proximal CRC Distal CRC Rectal CRC 0.60 ( ) ; (0.8.1) ; 0.53 N = ( ) ; ( ) ; 0.35 N = (0. 1.6) ; ( ) ; 0.1 IL1B 5 C>T IL1RN +2018T>C ( ); (0.6.); ( ) ; ( ) ; ( ) ; ( ) ; ( ); ( ); ( ) ; ( ) ; ( ) ; (0..56) ; 0.12 ) The subclassification of CRC by tumor site did not show any association with CRC for the SNPs studied. (table

12 CHAPTER 6. CONCLUSIONS Colorectal cancer occupies the first place among digestive neoplasms, with an increasing incidence in our country nowadays. The main treatment in CRC is represented by surgery, the response to oncological treatment being poor. Screening for colorectal cancer is another important matter, which may help discover the disease in an early stage when surgery can provide complet recovery. We emphasize the important role of anatomopathology department which is available to study the resection edges and the colorectal tumor. We studied some SNPs of IL1, one of the most important proinflammatory cytokines and we realised that the polymorphism IL1RN +2018T>C is associated with a high risk of developing colorectal cancer. We did not detect any efect of the other genetic variants of IL1B (IL1B 31T>C, IL1B 5C>T şi IL1B +395C>T) on the risk of CRC. A significant association has been observed for IL1RN +2018T>C polymorphism and CRC, the relative risk for carriers genotype was 2.2 (meaning these subjects are 2.2 times more exposed to develop the illness) when compared with the more frequent genotype. Another positive association was found for IL1RN genotype, that was limited to I and II stages. Concerning the other four studied cytokines (two of them having proinflammatory role IL8 and TNFα and the other two having antiinflammatory role ILR and IL10), we evaluated whether their polymorphism (one SNP for each interleukin: IL8 251T>A, TNFA 308G>A, ILR 3223C>T and IL A>G) influence the developing of colorectal cancer in Romanian population and we concluded that SNP IL A>G is associated with a lower risk of CRC A significant association was observed for IL A>G polymorphism: the subjects carrying AG genotype are protected against CRC (they have a 0.63 risk when compared with the more frequent genotype). Furthermore, in a dominant model the carriers of G allele were protected against CRC (these subjects have a 0.6 risk comparing with the same reference). In a stratified analyse according to tumoral grading, the only association between CRC and cytokine polymorphisms was found for carriers of IL G allele and was restricted to poorly differentiated cases. Our results are consistent with the hypothesis that higher production of IL10 in the colorectal mucosa would theoretically result in enhanced antiinflammatory response as well as reduced inflammation, finally leading to decreased CRC risk. For the other 3 studied SNPs (IL8 251T>A, ILR 3223C>T şi TNFA 308G>A) there was no statistical association with colorectal cancer. The results of our study may and must be considered a starting point in validating new biological markers, included in screening protocols for early detection, evaluating treatment and estimating the prognostic of colorectal cancer. 12

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