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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 2  |  Page : 54-57

CYP2C9 polymorphism is not associated with elevated carcinoembryonic antigen levels


1 Department of Surgery, Christchurch Hospital; University of Otago, Christchurch, New Zealand
2 Department of Surgical Sciences, Dunedin School of Medicine, University of Otago, Dunedin, New Zealand
3 Department of Biochemistry, University of Otago, Dunedin, New Zealand

Date of Web Publication27-Jun-2019

Correspondence Address:
Dr. Atanu Pal
Department of Surgery, Christchurch Hospital, University of Otago, Christchurch 8011
New Zealand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/WJCS.WJCS_39_18

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  Abstract 


Background: Carcinoembryonic antigen (CEA) is a glycoprotein that can be elevated in a number of benign and malignant conditions. In colorectal cancer, it is used as a prognostic marker and to detect recurrence. However, it lacks specificity and may become elevated in individuals without a history of cancer or other identifiable cause leading to costly and invasive investigation. Objective: The aim of this study was to assess whether genetic polymorphisms in the liver enzyme CYP2C9 could explain high CEA levels in otherwise normal individuals. Design: This is a case-control study. Setting: Individuals were genotyped for the poor metabolizer (PM) alleles CYP2C9*2 and CYP2C9*3 using predesigned TaqMan single nucleotide polymorphisms assays. Patients and Methods: Nineteen individuals with previously clinically unexplained elevated CEA and 567 healthy Caucasian controls were included. Main Outcome Measures: Chi-square analysis was used to test for association of CYP2C9 genotype with plasma CEA concentration. Sample Size: Nineteen individuals with previously clinically unexplained elevated CEA and 567 healthy Caucasian controls were included. Results: Fifteen of the 19 individuals with previously high CEA had elevated plasma CEA (>3.0μg/L) on re-testing. The frequency of CYP2C9 PM alleles in these 15 patients was not significantly higher than the frequency in controls. Conclusion: CEA concentrations do not appear to be influenced by CYP2C9 genotype, so this cannot be used to explain elevated CEA in the absence of an obvious clinical cause. Limitation: Small sample size.

Keywords: Carcinoembryonic antigen, colorectal cancer, CYP2C9 polymorphism, tumor marker


How to cite this article:
Hall C, Roberts R, Merriman TR, Pal A, Eglinton T, Wakeman C, Frizelle F. CYP2C9 polymorphism is not associated with elevated carcinoembryonic antigen levels. World J Colorectal Surg 2019;8:54-7

How to cite this URL:
Hall C, Roberts R, Merriman TR, Pal A, Eglinton T, Wakeman C, Frizelle F. CYP2C9 polymorphism is not associated with elevated carcinoembryonic antigen levels. World J Colorectal Surg [serial online] 2019 [cited 2019 Dec 10];8:54-7. Available from: http://www.wjcs.us.com/text.asp?2019/8/2/54/261545




  Introduction Top


Carcinoembryonic antigen(CEA) is a glycoprotein produced by the gastrointestinal(GI) tract during embryonic development that is not normally produced in significant quantities after birth. However, it is elevated in multiple pathologies. In clinical practice, CEA is most commonly used in colorectal cancer(CRC). While there is no evidence for using CEA concentration as a screening modality,[1] as it is often not elevated until the advanced disease is present,[1],[2],[3] CEA is used following diagnosis in CRC surveillance.[4],[5],[6],[7],[8],[9] Despite this many health-care practitioners still request a CEA if a patient presents with symptoms that are consistent with CRC, or indeed if there is a family history of CRC. This raises the dilemma of what should be done if a patient is found to have an elevated CEA but has no evidence of malignancy.

CEA undergoes approximately 90% hepatic “ first pass” metabolism.[10] CEA is initially taken up by the Kupffer cells where it is modified by removing the sialic acid residues. This form of CEA is then degraded by the liver parenchymal cells.[11] Cirrhosis, as well as other nonmalignant hepatic conditions, can lead to elevation in CEA due to alterations in first pass metabolism.[12] It is possible that loss-of-function genetic variants in first pass metabolism enzymes may also lead to elevated CEA levels in serum. CYP2C9 is a key liver enzyme.[13] The gene coding for CYP2C9 is highly polymorphic with over60 variants having been described(www.cypalleles.ki.se/). However, the vast majority of impaired CYP2C9-mediated metabolism can be explained by two poor metabolizer(PM) alleles CYP2C9*2 and CYP2C9*3. Both alleles carry loss-of-function single nucleotide polymorphisms(SNPs) which significantly slow the elimination of many common drugs. The CYP2C9*2 allele is present in 10–20% of Caucasian population but is rare in other ethnic groups.[13] This PM allele leads to a longer clearance time for warfarin, ibuprofen, and phenytoin. These drugs along with candesartan and celecoxib are also affected with the rarer CYP2C9*3 allele.

We hypothesize that individuals who carry the PM alleles CYP2C9*2 and / or CYP2C9*3 may show impaired metabolism of CEA thus resulting in increased CEA serum levels in the absence of pathology. To test this hypothesis, we genotyped CYP2C9*2 and CYP2C9*3 in 20 individuals with previously elevated CEA and 567 healthy controls with normal CEA.


  Patients and Methods Top


Study participants

Twenty Caucasians with clinically unexplained elevations in the CEA were recruited(patient group). Each individual had previously been referred to the colorectal service with an elevated CEA where no cause was found. Each had been thoroughly investigated following initial referral with gastroscopy, colonoscopy, and cross-sectional imaging comprising computed tomography(CT) of the chest, abdomen, and pelvis and/or positron emission tomography(PET)-CT. They were followed clinically for a minimum of 3years and had a repeat CT or PET-CT before the study to exclude new disease.

One patient was removed from the study due to inadequate follow-up time of 18months. 567 healthy Caucasians aged over19years and with no history of malignant or inflammatory disease were included as controls. The controls all had normal CEA.

All study participants gave their written informed consent, and ethical approval for this study was granted by the Upper South A Regional Ethics Committee ref: URA/10/EXP/056.

Measurement of carcinoembryonic antigen in serum

CEA was measured in serum using a chemiluminescent microparticle immunoassay(Abbott Park, Il 60064 USA). The serum sample and anti-CEA coated paramagnetic microparticles are combined to form a conjugate, which is then added to a propriety reaction mixture resulting in chemiluminescence.[14] The number of reactive light units emitted provides a direct measurement of the amount of CEA in the serum sample.

Canterbury District Health Board uses a reference range of 0–3ng/ml. Up to 11% of the local population is expected to have a CEA over3ng/ml without any pathological explanation. About 51.6% of these apparently normal individuals will be smokers;[14] so, 5.3% of the local population can be expected to have a raised CEA with no identifiable pathology and no history of smoking.

DNA extraction and CYP2C9 genotyping

Genomic DNA was collected from the peripheral blood of all study participants using guanidine isothiocyanate extraction and stored at−20°C in Tris-ethylenediaminetetraacetic acid buffer until analyzed. Apre-designed SNP TaqMan® assay(C__25625805_10 and C__27104892_10, Applied Biosystems, Carlsbad, California, USA) was used to perform genotyping for CYP2C9*2(rs1799853) and *3(rs1057910).[15] A 384-well plate was used with a total reaction volume of 5 μl and run on a Roche LightCycler® 480 real-time polymerase chain reaction System(Roche Diagnostics Corporation, Indianapolis, USA). Repeat analysis of 10% of samples, with concordance of 100%, confirmed the accuracy of the assay.


  Results Top


On re-testing of individuals with previously elevated CEA, serum CEA concentrations ranged from 1.2 to 12.6ng/ml (mean 5.4ng/ml), with 15 of 19 individuals still exhibiting elevated CEA concentrations(≥3ng/ml). The four patients with now normal CEA were removed from the study cohort. One of these patients was a current smoker, one an ex-smoker.

The patient group had a female preponderance of 2:1[Table1]. The age range was 45–78years with a mean age of 66.2. The CYP2C9 genotype frequencies for both patients and controls are shown in [Table2]. Homozygotes for CYP2C9*2 were higher in the patient population(6.7vs. 1.4%; P =0.05). However, the overall number of individuals expressing one or more of the PM alleles was similar(26.7vs. 31.9%; P =0.36) and so the significance of this is uncertain. Furthermore, there were no patients who expressed CYP2C9*3. This allele leads to a greater reduction of enzyme activity compared to CYP2C*2 and yet was found in the control population.{Table1}{Table2}


  Discussion Top


CEA has been intensively investigated with regards to its role in CRC. Recent large randomized controlled trials have shown that it is a useful tool during CRC surveillance.[16],[17],[18],[19] Moreover, multiple meta-analyses and a Cochrane review have demonstrated that inclusion of CEA as a part of multimodal follow-up can improve cancer-specific survival by discovering metastatic disease while it is still operable.[4],[5],[6],[7],[8]

Patients with a raised CEA are referred to the colorectal team, which initiates a range of investigations dependent on the patient's symptoms. This may include endoscopic evaluation of both the upper GI and lower GI tracts. Cross-sectional imaging is usually required in this patient population in addition to endoscopy to rule out any other cause of elevated CEA including small bowel tumors, hepatobiliary disease, and thyroid and ovarian tumors. This is especially true if the value is over10ng/mL as this is more likely to be associated with malignant disease.[9],[20],[21] If all investigations are normal, the patient often remains under investigation for several years until the benign nature of the CEA elevation is truly confirmed.

There have been multiple reasons postulated as to why some individuals have an elevated CEA in the absence of identifiable pathology, but to date, no causes have been determined. We hypothesized that the liver enzyme CYP2C9 might be involved as CEA has been shown to be elevated in a variety of benign hepatic conditions, particularly with hepatic obstruction such as in cholangitis secondary to common duct stones.[12] It is thought that the reason for this is multifactorial. First, any liver damage, albeit temporary, may reduce the efficiency of CEA metabolism; second, the obstructive pathology may also lead to reduced excretion with subsequent reduction in CEA once the biliary obstruction has resolved; and finally, the inflammatory process itself may also cause elevation in the circulating CEA. Nonmalignant liver disease tends to cause elevations in the region of 5–10ng/mL.[12] If the temporary liver damage is the primary reason for this increase in CEA then other abnormalities of liver metabolism, such as genetic changes within the cytochrome P450 system, may well explain why apparently normal individuals may have elevated CEA levels.

CYP2C9 was specifically chosen as it accounts for around 20% of the CYP protein in the adult liver[22] and genetic abnormalities, particularly CYP2C9*2 and CYP2C9*3 have been shown to lead to decreased metabolism in several drugs.[13],[22],[23] Heterozygotes for either of these PM alleles exhibit decreased metabolism of various drugs. In addition to this, there are studies which have looked at the association of elevated CEA with metabolic syndrome. Both high-fat diets and the subsequent manifestations of metabolic syndrome are thought to cause alterations in multiple components of the cytochrome P450 system.[23],[24],[25] It has been postulated that these changes ultimately result in insulin suppression, hypertension, dyslipidemia, and nonalcoholic fatty liver disease.[23] Kim etal.[24] reviewed 32,897 healthy Koreans and confirmed that higher CEA levels correlate with individuals who have features of metabolic syndrome. They hypothesized that the elevation may not simply be due to a change in the enzymes involved in hepatic metabolism but also due to increased levels of insulin and the low-level inflammatory states found in these individuals. Kang etal.[26] reviewed 18,131 healthy nonsmoking Koreans and found CEA level to be correlated with age, body mass index, white blood cell count, hemoglobin, aspartate transaminase, and HbA1c, and that adjusting for these variables increased the predictive value of CEA.

Our research did not confirm an association between CYP2C9 PM alleles and high serum CEA. Although there may be an association between high CEA and homozygosity for CYP2C9*2/*2, our sample size is too small to make any real inference. Yilmaz etal.[27] have also previously examined the relationship between the expression of CYP2C9 variants and serum tumor markers. They too were unable to find any significant difference in tumor marker expression in the varying genotypes associated with CYP2C9.


  Conclusion Top


High levels of CEA in otherwise normal patients lead to a lengthy and costly investigation process. False positives during CRC surveillance are equally as problematic. To date, an adequate physiological explanation for these high values does not exist. Our study provides preliminary evidence to suggest that a single enzyme pathway is not the reason for high CEA assays in the absence of pathology. However, further work is required.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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