Polymorphisms In Prostaglandin E2 (Pge2) Pathway Genes Alter The Risk For Colorectal Adenoma Recurrence After Polypectomy: A Chance For Individualized Surveillance?

Colorectal adenoma; colorectal adenoma recurrence; Genetic polymorphisms; TagSNPs; Cyclooxygenase-2; 15-Hydroxyprostaglandin Dehydrogenase; Multidrug-Resistance Protein 4; Prostaglandin Transporter

A hospital-based case-control study was conducted gathering 480 unscreened individuals and 195 patients diagnosed with colorectal adenomatous polyps to evaluate not only the influence of the genetic variability in four key genes of prostaglandin E2 (PGE2) pathway (COX-2/HPGD/SLCO2A1/ABCC4) in early stages of colorectal cancer development but also on the development of metachronous lesions. A total of 43 tagSNPs were characterized using the Sequenom platform through multiplexed amplification followed by mass-spectrometric product separation or allelic discrimination using real-time PCR.
Ten tagSNPs were identified as susceptibility biomarkers for the development of colorectal adenomas: the rs689466 in COX-2, the rs2555639, rs1346271, rs1863642 and rs12500316 in HPGD, the rs6439448 and rs1131598 in SLCO2A1 and rs9524821, rs1751051 and rs1678405 in ABCC4 genes. The individuals carrying the haplotype encompassing the rs9524821 and rs1751051 SNPs in ABCC4 gene presented a risk of 3.9 for adenomas development (95%CI:2.28-6.65, P<0.001). Furthermore, the best four-locus gene-gene interaction model included the rs1346271, rs186342 and rs12500316 SNPs in HPGD and rs1678405 in ABCC4 genes and was associated with a 13-fold increased susceptibility (95%CI:3.84-46.3, P<0.0001, cross-validation (CV) accuracy: 0.78 and CV consistency: 8/10). Interesting, in high-risk patients the rs1678405 ABCC4 SNP had a lower hazard ratio (HR) and half the crude risk for adenoma recurrence at 36 months, when comparing with the overall high-risk patients (7% vs 14%).
Genetic polymorphisms in the COX-2/PGE2 pathway appear to contribute to the development of colorectal adenomas and influence the interval time to adenomas recurrence. A deeper knowledge on CRC etiology through the identification of biomarkers of colorectal carcinogenesis might allow a better definition of risk models that are more likely to benefit from targeted preventive strategies to reduce CRC burden.

Colorectal cancer (CRC) is the third most common malignancy in men, and the second in women in developed regions, with a lifetime risk of 4.3% and 2.7%, respectively] [GLOBOCAN 14]. According to the International Agency for Research on Cancer (IACR) estimates, 837.437 individuals will be diagnosed and 381.188 will die from CRC in 2020, a 13.6% and 14.4% increase in this cancer burden and mortality from previous estimates [GLOBOCAN 14].
Colorectal adenomatous polyps are well-characterized CRC precursors []. Although most adenomas are asymptomatic and do not progress into cancer, the majority of CRC will develop through the adenoma-cancer sequence on an average of 10-15 years []. Approximately one-third of people will develop at least one adenoma by the age of 60 years [].
CRC screening has been shown to reduce the incidence and CRC mortality through the endoscopic detection and removal of the precancerous lesions []. Still, these patients are at increased risk for developing metachronous colorectal adenomas or even cancer, with the recurrence rate being around 40-50% []. Despite population-based screening being widely recommended in Europe, the development of primary prevention strategies is an important goal to reduce the risk of colorectal neoplasia, considering the inherent limitations of colorectal screening and adherence rates [].
Nonsteroidal anti-inflammatory drugs (NSAIDs) is the most widely studied pharmacological agent in colorectal cancer prevention and its use reduces the occurrence of advanced adenomas by 28% [and the recurrence by 34%, mainly by targeting the cyclooxygenase-2 enzyme (COX-2) [].
Up-regulation of COX-2 is an early and key event in colorectal carcinogenesis []. Deregulation of COX-2 expression, observed in half of adenomatous polyps, leads to an increased biosynthesis of prostaglandin E2 (PGE2) []. The pleiotropic effects of higher levels of PGE2 contribute to key steps of cancer development including stimulation of cell proliferation, angiogenesis, invasiveness and migration, inhibition of apoptosis and immunosurveillance []. The degradation of PG is mediated by the NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) encoded by the hydroxyprostaglandin dehydrogenase (HPGD) gene that directly counteracts the COX-2 oncogenic PGE2 pathway []. The expression and activity of 15-PGDH is repressed in colorectal cancer and Apcmin mouse adenomas [] Furthermore, low levels of rectal 15-PGDH were associated with increased adenoma recurrence []. The multidrug resistance-associated protein 4 (MRP4), encoded by the ATP-binding cassete sub-family C member 4 (ABCC4) gene, and the prostaglandin transporter (PGT), encoded by the solute carrier organic anion transporter family, member 2A1 (SLCO2A1) gene, are the specific prostaglandin membrane transporters that regulate PGE2 levels in the extracellular microenvironment []. The MRP4 exports the newly synthesized PGE2 to the extracellular milleu where a plethora of pathways will be activated through binding to specific G-protein couple receptors (EP1-EP4) []. The clearance of PG from circulation for subsequent 15-PGDH inactivation is mediated by PGT []. PGT and MRP4 mRNA levels were reported to be inversely regulated in human CRC, with PGT expression being repressed and MRP4 up-regulated in CRC tissues and cell lines leading to higher levels of PGE2 extracellularly thus potentiating the effects of COX-2/PGE2 pathway.
The genetic background certainly plays a key role in CRC development, as it is expected to explain 35% of familiarity []. Considering that the aforementioned genes are not only highly polymorphic but their expression span several folds, one could hypothesize that an unbalance in PGE2 levels reflecting potential functional polymorphisms might influence colorectal carcinogenesis and consequently the genetic susceptibility for the development of colorectal precancerous lesions.
Using a tagSNP approach, our group recently reported the involvement of several polymorphisms in COX-2/HPGD/SLCO2A1/ABCC4 genes on CRC development []. So, with this study we aimed to investigate whether the same panel of tagSNPs was also associated with earlier stages of colorectal tumor development. Furthermore and to our knowledge this is the first study to evaluate the influence of polymorphisms in HPGD, SLCO2A1 and ABCC4 genes in the occurrence of colorectal metachronous lesions.

Type of study
A hospital-based case-control study was design involving a group of unscreened individuals (control population) and patients diagnosed with colorectal adenomas (case population) to address the role of polymorphisms in PGE2 pathway genes on the risk of colorectal adenoma onset. These patients were further stratified according to the presence of metachronous lesions in a retrospective case-cohort study. All individuals were from the northern region of Portugal and recruited at the Instituto Portugu??s de Oncologia do Porto (IPOP) or Centro Hospitalar do Porto (CHP). This project was approved by the Ethics Committee of both institutes (ref. 0084/08 and ref. 080 DEFI/ 100-CES, respectively) and Comiss??o Nacional de Protec'?o de Dados (ref. 6619/2011), the Portuguese Data Protection Authority.
Control group
Unscreened individuals between 50 and 75 years of age, without any clinical evidence of CRC or other oncologic malignancy were randomly recruited from the blood donor's service at IPO-Porto between July 2005 and February 2008.
CRC patients group
Patients diagnosed with one or more adenomas between 1996 and 2008 were enrolled in this study after reviewing a colonoscopy database from the Gastroenterology departments at IPOP and CHP. The inclusion criteria were: (1) age between 50 to 75 years; (2) with a total colonoscopy with good to excellent preparation at diagnosis; (3) without history of inflammatory bowel disease or family history of colorectal tumors; and (4) without previous diagnose of CRC.
Patients with personal history of colorectal adenomas were further categorized according to the recurrence status. Adenoma recurrence was defined as the diagnosis of an adenoma after having a normal total colonoscopy, with a good to excellent preparation, at least one year after the initial diagnosis of adenoma.
Nearly three thousand individuals had history of adenomas, although only less than 10% complied with the inclusion criteria. A total of 256 patients were included in this study. From these we were only able to obtain DNA samples from 195 patients. No differences were observed between demographic variables, lifestyle habits and tumor characteristics between these patients and the overall population of patients.
Medical records were reviewed to extract the clinicopathological variables and to exclude misclassification bias. During telephone interviews patients were asked to recall their lifestyle habits (smoking habits, etc) in the years preceding their diagnosis.
Sample collection and biological processing
The DNA was extracted from formalin fixed paraffin embedded (FFPE) blocks from the Pathology Departments at both centers, using the GRS Genomic DNA Kit ' Tissue, in accordance with the manufacturer's protocol (GRiSP, Porto, Portugal).
DNA was quantified using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and stored at -20??C until genotype examination. The DNA quality was determined by measuring the optical density (OD) 260/280 ratio.
Polymorphisms selection
The strategy for polymorphisms selection has been described elsewhere []. Briefly, 55 tagSNPs were included after being retrieved from a set of common SNPs in the Caucasian population of HapMap project (CEU) (1) with minor allele frequency equal or superior to 15%; (2) within the coding region of the genes plus 2Kb upstream and downstream; (3) with a r2 superior to 0.8 and (4) that successfully converted to the Sequenom platform.
Furthermore, the rs20417, rs689466 and rs5275 polymorphisms in COX-2 and rs2612656 and rs2555639 in HPGD genes, that were previously associated with colorectal tumors development, were also included [].
Genotype characterization
TagSNP genotyping was performed using MassARRAY iPLEX Gold technology (Sequenom, San Diego, CA) based on multiplexed amplification followed by mass-spectrometric product separation. This technique was carried-out by the Unidade de Gen??mica/Servi??o de Genotipagem do Instituto Gulbenkian de Ci??ncia.
The rs689466, rs20417, rs5275, rs2612656 and rs2555639 were characterized through allelic discrimination (Real-Time Polymerase Chain Reaction) using validated TaqMan?? SNP genotyping assays (C___2517145_20, C___7550203_10, C___15909858_20, C___16038735_10, respectively) with the exception of the rs20417 SNP which was custom designed (Applied Biosystems, Foster City, California USA). Allelic discrimination was performed by measuring end-point fluorescence using ABI PRISM Sequence Detection System (Applied Biosystems, Foster City, California USA).
Quality control
Genotypes were excluded from the analysis if any of the following criteria was applied: call rate inferior to 0.90; concordance rate inferior to 0.95 and Hardy-Weinberg equilibrium (HWE) with P<0.05. Blank templates were included in each 96 and 384-well plates to ensure contamination-free results. Two researchers performed the genotype interpretation independently and five to ten percent of all samples were randomly selected and re-submitted to a new genetic characterization to confirm the genotypes. Furthermore, the use of FFPE samples for SNP genotyping was previously validated by comparing the genotypes from 20 DNAs isolated from fresh peripheral blood and paired FFPE samples from CRC patients.

Statistical analysis
For genetic distribution analysis, the Hardy'Weinberg equilibrium was tested by the Pearson's goodness-of-fit test to compare the observed versus the expected genotype distribution among the control population.
Data analysis was performed using the computer software IBM Statistical Package for Social Sciences-SPSS (IBM Corp., Armonk, New York, USA) for Macintosh (version 19.0). Chi-square analysis was used to compare categorical variables, using a 5% level of significance. Non-parametric tests were used to compare mean values. Odds ratio (OR) and its 95% confidence interval (CI) were calculated as a measure of the association between the genetic variants and the risk for the development of CRC. Covariates expected to be involved on colorectal carcinogenesis were included in the logistic regression analysis. The false positive report probability (FPRP) was used to confirm the noteworthiness of significant findings on the overall risk for colorectal adenoma development, according to the study by Wacholder and colleagues [23]. The FPRP threshold was set at 0.5 under an assigned prior probability ranging from 0.01 to 0.10 to detect an OR of 1.5.
Haplotype analysis was performed at a gene level using the SNPStats software (www. http://bioinfo.iconcologia.net/SNPstats). The haplotype frequencies were estimated using the implementation of the EM algorithm coded into the haplo.stats package. The most frequent haplotype was automatically selected as the reference category. After excluding the genetic variations most likely to represent false positive findings, all polymorphisms with significant associations were included within each gene.
The open-source multifactor dimensionality reduction (MDR) software (version 3.0.2) (www.epistasis.org) was used to assess potential gene-gene interactions between SNPs with statistical significant impact on CRC genetic susceptibility. The fitness of an MDR model was estimated by determining the testing accuracy and its cross-validation consistency (CVC). Using a 10-fold cross-validation method the data was divided into 10 sets, in which 9 subsets were training sets and one subset was a test set. Hence, the CVC is a measure of the number of times of 10 divisions of the dataset the best model was extracted. The single best model normally has the maximal testing accuracy and CVC. Statistical significance was evaluated using a 1000-fold permutation test to compare observed testing accuracies with those expected under the null hypothesis of null association. Permutation testing corrected for multiple testing by repeating the entire analysis on 1000 datasets that were consistent with the null hypothesis.
Kaplan-Meier curves were used to evaluate the correlation between the genetic variants and time to recurrence; log rank statistical test was used for curves comparison. '
Study population
A description of the population understudied is displayed in Table 1 and as can be observed patients with history of adenomas were slightly older than controls' (61 vs 58, P<0.001). Males and non-smokers were overrepresented in either group (57 vs 65%, P=0159 and 72 vs 70%, P=0.075, respectively).
In over 70% of patients, less than three adenomas (71%) were detected. Most were located distally to the splenic flexure (82%) and were larger or equal to 10 mm in size (64%). Histologically, high-grade dysplasia was described in 33% of index adenomatous polyps.
High risk patients for adenoma recurrence (adenoma with villous histology or high grade dysplasia or ' 10 mm in size, or ' 3 adenomas) represented 72% of cases' population. The median follow up time was 76 months (22-201) and metachronous lesions were identified in 46% of patients with personal history of adenomas. No differences were observed between these patients and the ones without adenoma recurrence during the follow up period.
Risk estimates for colorectal adenoma
Eight SNPs were excluded from the analysis due to genotyping failure and four SNPs were dropped because their frequencies deviated from HWE (P<0.05). A total of 43 SNPs were included in the risk estimate analysis. The mean genotype call and concordance rates were 99.02% and 99.3%, respectively. The description of selected SNPs is displayed in Table S1 of supporting information.
Using the false positive report probability (FPRP), nine polymorphisms retained their association with colorectal adenoma development, as observed in Table 2. Different prior probability values were considered depending on the availability of previous functional or epidemiologic data.
Individuals homozygous for the minor G allele of the rs689466 SNP in COX-2 gene were overrepresented in the group of cases (10% vs 3%, in controls) leading to a 3-fold increased risk for colorectal adenoma (95%CI:1.52-6.86, P=0.002) in the multivariate analysis, including age, sex, smoking habits and hospital center as covariates. Following a recessive model, the rs2555639 polymorphism was positively linked with the onset of colorectal precancerous lesions (OR=2.48; 95%CI:1.36-4.53, P=0.003), whereas the rs1346271, rs1863642 and rs12500316 genetic variants in the HPGD gene were associated with a 45-51% protection in carriers of the minor alleles (OR=0.55; 95%CI:0.35-0.85, P=0.008, OR=0.55; 95%CI:0.36-0.85, P=0.007 and OR=0.49; 95%CI:0.31-0.78, P=0.002 for the rs1346271, rs1863642 and rs12500316, respectively). Stressing the SLCO2A1 gene, individuals carrying the rs6439448G allele presented a 62% decreased risk for colorectal adenomas (95%CI:0.22-0.65, P<0.001). Although with a lower influence, the rs1131598 was also inversely associated with colorectal adenomas development (OR=0.58; 95%CI:0.41-0.84, P=0.018). Three out of the 21 polymorphisms in ABCC4 gene appeared to have an impact in colorectal carcinogenesis in early stages. The AA and CC genotypes of rs9524821 and rs1751051 polymorphisms, more than double the susceptibility for colonic precancerous tumors (OR=2.38; 95%CI:1.39-4.09, P=0.002 and OR=2.75; 95%CI:1.58-4.80, P<0.001). In contrast, a protective role was noticed for genotypes carrying the rs1678405C allele SNP was noticed, even more in homozygous for the C allele (OR=0.41; 95%CI:0.27-0.63, P<0.001).
Haplotype analysis for colorectal adenomas
Since multiple SNPs were addressed within each gene and multiple associations were reported, with exception of COX-2 gene, a haplotype analysis was performed. The frequencies of derived haplotypes from HPGD, SLCO2A1 and ABCC4 genes are presented in Table 3. The most frequent haplotype of the HPGD gene, the CGGT, was present in 35% of controls and used as the reference one. The blocks containing the rs1250016T allele, TGGT, and the rs1346271C-rs1863642T-rs2555639C alleles, CCTC, were associated with a high protection, although they were detected roughly in 1% of cases (OR=0.05; 95%CI:0.01-0.15, P<0.001 and OR=0.06; 95%CI:0.01-0.33, P=0.001, respectively). Consistent with the individual SNP analyses, the haplotypes carrying the rs1131598G allele, GC, or rs6439448A allele, AA, displayed a protective role for the development of colorectal adenoma (vs AC, OR=0.68; 95%CI:0.49-0.95, P=0.024 and OR=0.50; 95%CI:0.33-0.76, P=0.001, respectively). No block containing both the minor alleles was present at a frequency higher or equal to 5%. Out of the six haplotypes described for the ABCC4 gene, the block carrying the rs9524821A and rs1751051A alleles boosted even further the susceptibility for colorectal precancerous lesion reported in the individual analysis in contrast with the GTT haplotype (OR=3.90; 95%CI: 2.28-6.65, P<0.001).
Gene-gene interaction analysis in colorectal adenoma

To address possible interactions between the noteworthy SNPs from the main analysis, an exhaustive MDR approach was employed and Table 4 summarizes the best interactive models obtained. All best models from one to four-locus were significant at P'0.001, and the highest cross-validation consistency was observed with the two-factor interaction model (9/10). Nevertheless, the best four-locus model achieved the highest testing accuracy of 78% for predicting the development of colorectal adenomas, although with a lower cross-validation consistency (8/10). This interaction model included the rs1346271, rs1863642 and rs12500316 polymorphisms in HPGD gene and rs1678405 in ABCC4 gene and was associated with a 13-fold increased adenoma risk (95%CI: 3.84-46.3, P<0.0001).
Risk assessment for the development of colorectal metachronous lesions
The genetic variability in COX-2 and HPGD genes does not appear to contribute to the development of metachronous tumors in patients previously diagnosed with colorectal tumors, as observed in Table 5. Three polymorphisms in SLCO2A1 gene were reported to influence the susceptibility for colorectal adenomas recurrence. The rs1131598GG homozygous genotype was associated with an enhanced risk of 6.2 (95%CI:1.29-29.5, P=0.023) and rs9820625C allele carriers with a nearly three-fold increased predisposition (95%CI: 1.08-6.29, P=0.033). On the other hand, individuals carrying the rs7340717T allele had a 51% protection for developing metachronous adenomas (95%CI:0.20-1.00, P=0.049). Under a dominant model of inheritance, the rs1751031 and rs9524821 polymorphisms in ABCC4 gene appear to display a protective behavior (OR=0.30; 95%CI:0.12-0.75, P=0.010 and OR=0.40; 95%CI:0.18-0.89, P=0.024, respectively), in contrast with the positive association observed in the presence of rs8002180C allele (OR=2.17; 95%CI:1.01-4.66, P=0.047). None of the aforementioned SNPs retained their noteworthy upon the FPRP analysis, potentially indicating false positive findings (FPRP>0.5).
Influence on the time to recurrence and crude risk
We next, inquired if polymorphisms in these key genes in PGE2 pathway could influence not only the time but also the crude risk for adenomas recurrence at 36, 60 and 120 months, following the recommendations for post-polypectomy colonoscopy surveillance [] (Table 6). Although no difference was observed on the time to adenoma recurrence (112 vs 105 months, P=0.788) or recurrence rate (46%, P=0.996) between the high and low risk patients, 14% of all adenomatous polyps recurred at 36 in the high risk group in contrast to the 2% reported in low risk patients. Additionally, nearly 95% (18/19) of metachronous advanced adenomas were described in the high risk group, with 28% and 67% being diagnosed at 36 and 60 months (data not shown).
The contribution of the genetic background appeared to be particularly relevant on patients at low risk. For example, the rs9524821AA genotype not only was associated with a nearly three-fold increased susceptibility in the cox regression analysis (95%CI:1.07-8.03), but also half of patients carrying this genotype had adenoma recurrence at 60 months, considerable higher than the 21% noticed in low risk patients. Similarly, patients' carriers of rs2274403AA genotype had a lower interval until recurrence (85 (29-140) vs 122 (109-135)) with 44% of metachronous tumors developing by 36 months (vs 23% for AG/GG). In the high risk group the genetic polymorphism with potential for influencing current guidelines is the rs1678405, for which TT carriers had a lower hazard ratio, with a higher time to recurrence (109 (89-129) vs 90 (76-104)) and half the crude risk for recurrence at 36 months, in comparison with the overall high risk patients (7% vs 14%).


Colorectal cancer still remains a major clinical and public health challenge that could be prevented by applying the current knowledge about CRC prevention and improving the adherence to established screening guidelines [].
The rapid decline on CRC incidence over the past decade has been largely attributed to the endoscopic detection and removal of precancerous adenomatous polyps and endoscopic follow up of these patients with personal history of colorectal adenomas []. Nevertheless, not only are the compliance rates far from the desirable, even in countries with implemented population-based CRC screening guidelines, also important lesions are missed or incompletely removed during colonoscopy [].
The search for susceptibility biomarkers in colorectal carcinogenesis might reveal an important tool to select unscreened individuals to CRC screening or even to complementary chemopreventive strategies with NSAIDs by allowing the identification of individuals at higher risk for the development of colorectal tumors. Currently, NSAIDs use in CRC prevention is hampered by the adverse gastrointestinal side effects associated with its regular use in average risk populations [].
The efflux-dominated flow of PG during carcinogenesis as a reflection of an increased expression of COX-2 and MRP4 and down regulation of 15-PGDH and PGT leads to an accumulation of PGE2 in the extracellular milieu culminating in the activation of a plethora of pathways that stimulate tumor development [].
In the present study, we addressed the role of 43 tagSNPS in four candidate genes (COX-2/HPGD/SLCO2A1/ABCC4) of COX-2/PGE2 pathway on the development and recurrence of colorectal adenomatous polyps in a Northern Portuguese population. Recently, using the same tagSNPs approach and targeting the same pathway we also identified the rs689466A>G polymorphism in COX-2, the rs1346271G>C in HPGD, the rs6439448C>A in SLCO2A1 and the rs1751051T>A in ABCC4 genes polymorphisms as susceptibility biomarkers for CRC, supporting the associations reported here and the role they might portray in colorectal carcinogenesis [].
The homozygous GG genotype for the rs689466 SNP, also known as -1195A>G COX-2 polymorphism, associated presently with a three-fold higher predisposition, was previously related with a higher risk for duodenal adenomatosis in patients with familial adenomatous polyposis (FAP) []. Although representing a hereditary syndrome, deregulation of COX-2 expression was observed in normal and duodenal adenomas of FAP patients []. Furthermore, our group in a earlier study, observed a higher transcriptional activity in HCT116 and HCA-7 CRC cell lines transfected with COX-2 promoters' encompassing the rs689466G allele, thus providing a biological plausibility for the epidemiologic observations [].
Thompson and colleagues [], first associated the rs2555639T>C SNP located at 17.74kb upstream the 5'UTR of HPGD gene with a 40% increased risk for CRC in TT homozygous carriers. Surprisingly, in our population this SNP not only appears to be more relevant in early stages of colorectal carcinogenesis, but the opposing rs2555639CC genotype was the one linked to colorectal adenomas onset. This conflicting data, might reflect population stratification involving different genetic ancestry, considering that the initial study involved participants from the Kentucky Surveillance, Epidemiology and End Results (SEER) registry most likely with Northern or Western European ancestry (English, German, Irish ancestry).
Furthermore, the rs1346271G>C, rs1863642G>T and rs12500316C>T tagSNPs in HPGD gene displayed a protective role in colorectal adenoma onset, as reported by Edwards and colleagues []. Apart from the rs1346271 polymorphism, that was previously associated with a reduced risk for CRC and locates in HPGD promoter region altering the binding site for nuclear proteins (SNPinfo software), no other SNP provides a biological reasoning for the protection observed [].
The PGT and MRP4 specific PG membrane transporters are encoded by highly polymorphic genes []. Still, the study of genetic variants in SLCO2A1 and ABCC4 genes on the etiology of malignant diseases has been rather neglected [meu e o estudo em CCR].
In our population, two polymorphisms in SLCO2A1 gene appeared to modulate the susceptibility for colorectal adenoma (rs1131598A>G and rs6439448C>A), although it was more noticeable in rs6439448A allele carriers, in whom a 60% protection was observed. Biologically, the rs6439448 SNP tags two other polymorphisms with predicted impact on PGT expression: the rs2370512T>A located in the 3'UTR could affect the binding of microRNAs and stability of mRNA and the nonsynonymous rs34550074G>A SNP at codon 396 codes for two different amino acids (Ala396Thr) with potential repercussion on protein structure and function. Similarly, the rs1131598A>G polymorphism, located at 3'UTR, is predicted to influence mRNA stability and thus the PGT protein expression.
Remarkably, homozygous mutations in HPGD and more recently in SLCO2A1 gene were identified as causative agents for the development of primary hypertrophic osteoarthropathy (PHO) []. Similarly to neoplastic tumor genesis, increased levels of PGE2 play a role in the pathogenesis of PHO, thus reinforcing the impact that genetic variability in these genes might portray in disease development by disrupting the normal 15-PGDH and PGT levels or activity [].
Regarding the ABCC4 gene, three polymorphisms influenced the risk for adenoma onset (rs1678405, rs9524821 and rs1751051). More interesting, individuals carrying the haplotype containing both the A alleles for the rs9524821 and rs1751051 SNPs had a nearly 4-fold increased susceptibility. The in silico analysis did not provide any biological clue for the involvement of these polymorphisms in MRP4 expression or function.
Common diseases are believed to develop from the combined effect of low penetrance genes. The gene-gene interaction analysis reinforced the data from the single-locus analysis lending further support to the involvement of genetic susceptibility biomarkers in colorectal carcinogenesis.
The current post-polypectomy guidelines recommend endoscopic surveillance based on risk stratification upon the endoscopic findings at baseline colonoscopy []. For individuals at low risk of adenoma recurrence (1-2 tubular adenomas <10 mm in size with low grade dysplasia) a repetition of colonoscopy 10 years after the index colonoscopy is suggested. In the high risk group (patients with adenomas with villous histology or high grade dysplasia or ' 10 mm in size, or ' 3 adenomas) a surveillance colonoscopy 3 years after the baseline colonoscopy is recommended.
In this study we observed that polymorphisms in the COX-2/PGE2 pathway, particularly on the ABCC4 gene, influenced not only the hazard ratio for the development of metachronous adenomas, but perhaps more importantly the probability for recurrence considering the surveillance intervals currently recommended. As an example, the individuals carrying the rs9524821AA genotype in the low risk group presented a nearly three-fold higher hazard ratio for adenoma recurrence and nearly half of them developed metachronous lesions by 60 months (vs 16%, for G allele carriers). In contrast, the rs1678405TT genotype was associated with a 7% recurrence at 36 months in the high risk group, in opposition to C allele carriers (23%). Considering the limited number of participants in this study we were not able to specifically assess the influence of these SNPs on the risk for the development of advanced colorectal adenomas, but assuming an equal distribution among genotypes, this could represent that patients carrying the rs1678405TT genotype might benefit from a looser surveillance interval.
These observations should be interpreted with caution considering the several drawbacks encountered in this study. Inherent to a retrospective study we cannot rule out selection bias, even more considering that our control population was represented by unscreened individuals. Nevertheless, if this were true we would expect stronger associations; or recall bias that could decrease the availability and accuracy of collected data, compromising our ability to estimate possible gene-environment interactions. Our major limitation certainly falls on the low statistical power (<80%), namely when carrying out the analysis on the recurrence of adenomas. Considering the colonoscopy missing rate, that can reach 26% in polyps <5 mm, and the incomplete removal of lesions during baseline colonoscopy we imposed strict inclusion criteria to ensure the correct classification of patients with previous history of adenoma []. Replication studies with larger and independent populations are needed to clarify the involvement of these polymorphisms in early stages of colorectal carcinogenesis. Furthermore, functional studies evaluating the repercussion of the aforementioned SNPs on protein expression/function will allow a deeper understanding of their real contribution on cancer development.
In this study, we observed the involvement of several polymorphisms in COX-2/HPGD/SLCO2A1/ABCC4 genes in colorectal adenoma development and recurrence. Furthermore, the incorporation of genetic variants in current risk models might provide a better balance between benefits and drawbacks of post-polypectomy surveillance.

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