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Year : 2019  |  Volume : 56  |  Issue : 2  |  Page : 130--134

Association of serum level of vitamin D and VDR polymorphism Fok1 with the risk or survival of pancreatic cancer in Egyptian population

Amal Ahmed Mohamed1, Ahmed Moustafa Aref2, Soha M Talima3, Reham A A Elshimy4, Shawkat S Gerges5, Mohamed Meghed5, Fawkia Eissa Zahran6, Eman H EL-Adawy7, Sherief Abd-Elsalam8,  
1 Department of Biochemistry and Molecular Biology, National Hepatology and Tropical Medicine Research institute, Nasr City, Cairo, Egypt
2 Faculty of Biotechnology, October University for Modern Sciences and Arts (MSA), 6th October City, Giza City, Giza, Egypt
3 Department of Clinical Oncology, Kasr Al-Ainy Center of Clinical Oncology and Nuclear Medicine (NEMROCK), Kasr Al-Ainy School of Medicine, Cairo University, Giza City, Giza, Egypt
4 Department of Clinical Pathology, National Cancer Institute, Cairo University, Cairo Governorate, Mansoura City, Egypt
5 Department of Surgery, National Hepatology and Tropical Medicine Research institute, Nasr City, Cairo, Egypt
6 Department of Internal Medicine, Faculty of Medicine (Girls), Al-Azhar University, Nasr City, Cairo, Egypt
7 Department of Internal Medicine and Endocrinology, Faculty of Medicine, Mansoura University, Elgomhouria St., Mansoura City, Egypt
8 Department of Tropical Medicine, Faculty of Medicine, Tanta University, l-Gaish, Tanta Qism 2, Tanta, Gharbia Governorate, Egypt

Correspondence Address:
Sherief Abd-Elsalam
Department of Tropical Medicine, Faculty of Medicine, Tanta University, l-Gaish, Tanta Qism 2, Tanta, Gharbia Governorate
Egypt

Abstract

BACKGROUND AND AIMS: Pancreatic cancer (PC) is the fourth most common cause of death from cancer in Egypt. Few studies have been conducted to assess the relationship between vitamin D serum level and vitamin D receptor (VDR) polymorphisms with the survival of PC patients. This is the first study in Egypt to investigate the association of the status of vitamin D serum level and genotypic distribution of single nucleotide polymorphisms (SNP) Fok1 with the risk of developing PC and whether they could detect survival or not. PATIENTS AND METHODS: The study included a total of 47 PC cases that were histopathologically proven to have PC, and 37 controls that were attending at the same time for investigation but proved that they were all PC free. Pre-diagnostic concentrations of vitamin D and VDR polymorphism Fok1 were assessed from all participants in the study. RESULTS: There was a 1.5-fold increase in the serum level of vitamin D in PC patients when compared to non-PC subjects. Regarding VDR Fok1, polymorphism distribution in PC was CC (Wild Type) 26 (55.3%), CT 16 (34%), and TT 5 patients (10.7%). For the control group, CC was found in 24 (64.8%), CT in 12 (32.4%), and TT genotype was found only in one individual 1 (2.8%) with no statistically significant difference between the two studied groups (P 0.72). CONCLUSION: Low serum vitamin D or VDR-SNP is not a risk factor for PC in Egyptian patients. Recommendations to increase vitamin D concentrations in healthy persons for the prevention of cancer and improving overall survival should be carefully considered.



How to cite this article:
Mohamed AA, Aref AM, Talima SM, A Elshimy RA, Gerges SS, Meghed M, Zahran FE, EL-Adawy EH, Abd-Elsalam S. Association of serum level of vitamin D and VDR polymorphism Fok1 with the risk or survival of pancreatic cancer in Egyptian population.Indian J Cancer 2019;56:130-134


How to cite this URL:
Mohamed AA, Aref AM, Talima SM, A Elshimy RA, Gerges SS, Meghed M, Zahran FE, EL-Adawy EH, Abd-Elsalam S. Association of serum level of vitamin D and VDR polymorphism Fok1 with the risk or survival of pancreatic cancer in Egyptian population. Indian J Cancer [serial online] 2019 [cited 2019 Sep 20 ];56:130-134
Available from: http://www.indianjcancer.com/text.asp?2019/56/2/130/257549


Full Text



 Introduction



Pancreatic cancer (PC) is the fourth most common cause of death from cancer in the USA and expected to be the second deadliest cancer in the USA by 2020.[1] Prognosis is very poor; 1 and 5 years survival rates are 25% and 4%, respectively. As most patients are diagnosed at stage 3, good numbers of patients die within 1 year from its discovery.[2]

In Egypt, PC accounts for about 2% of all cancers, with an age-adjusted incidence of 3.2/100,000, which varies from lower, middle, and upper Egypt with incidence rates of 3.2%, 1.94, and 3.6%, respectively.[3]

There is no effective tool for diagnosis, and the exact etiologies are still unknown; cigarettes smoking, high blood glucose level, and chronic pancreatitis are known risk factors for PC.[4]

The results of obesity and physical activity are controversial; many results have identified obesity as a risk factor,[5] whereas other studies did not recognize that lack of physical activity and high body mass index (BMI) are risk factors for PC.[6] In addition, high processed meat intake may be another risk factor for PC, but this is probably secondary to carcinogenic substances evolved during meat processing.[7]

Because PC is very resistant to treatment, there are efforts to find protective factors. Vitamin D seems to prevent many types of cancers, not only for PC but also can prevent colon, ovary, and breast cancer.[8]

The main circulating form of vitamin D in humans is 25-Hydroxyvitamin D (25(OH)D), and it is considered the best for assessment of vitamin D status derived from both sun exposure and diet. Vitamin D has many biological functions and can prevent malignant transformation as it has an impact on cell division, differentiation, adhesion, and apoptosis as it regulates the levels of p21 and p27, which in turn control the cell cycle. In addition, the tumor suppressor gene p53 that is activated in case of DNA damage is regulated by vitamin D.[9],[10]

Studies examining dietary vitamin D or measured 25(OH)D concentrations and PC have shown conflicting results. Many reports have demonstrated reduced risk of PC with high vitamin D levels[11], whereas others has shown statistically significant 3-fold increase in the risk of PC with higher vitamin D levels.[12]

Studies have linked possible association between different types of cancers and several single nucleotide polymorphisms (SNPs) in the vitamin D receptor (VDR), which may interfere with its function, thus increasing risk of cancer.[13] Approximately, 200 VDR-SNPs have been identified, however, only Fok1, BsmI, TaqI, ApaI, EcoRV, and Cdx2 were frequently associated with tumorigenesis.[13]

SNP rs2228570 is a T/C substitution located 10 base pairs upstream contains a Fok1 cleavage site and an alternative upstream translation start codon, which leads to the production of a different VDR protein with three amino acids longer than normal that was found to be less transcriptionally active.[14]

Few studies have been conducted to assess the relationship between vitamin D serum level and VDR polymorphisms with the survival of PC patients.[15]

To our knowledge, this is the first study in Egypt to investigate the status of vitamin D serum level and genotypic distribution of SNP Fok1 and the risk of developing PC and whether they could detect survival or not.

 Patients and Methods



The present study was conducted according to the guidelines of the ethical principles outlined in the declaration of Helsinki. The study was approved by the ethical committee of Tanta University Faculty of Medicine. This study was a multi-center study that was conducted in National Hepatology and Tropical Medicine Research, National Cancer Institute, Clinical Oncology Department-Cairo University, October University for Modern Sciences and Arts and Tropical Medicine Department, Tanta University. The study included a total of 47 histopathologically proved PC cases, and 37 controls that attended for investigation at the same time, but proved to be all pancreatic cancer - free.

All participants in this study were questionnaired on general background characteristics including medical background, history of PC, occupation, residency, meat consumption, alcohol consumption, coffee drinking, smoking, and if they had abdominal pain or elevated body temperature or not. Overall survival time was calculated from the date of cancer diagnosis until the date of death or last follow-up if the patient was still alive. Deaths of those patients were confirmed from next-of-kin.

Sample collection and biochemical analysis

None of the patients had received chemotherapy or radiotherapy prior to serum collection. Ten mL of venous blood was taken in dry sterile containers. After centrifugation, portions of blood were allowed to clot and then centrifuged at 3500 g for 10 min to separate the serum designed for assessment of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transpeptidase (GGT) activities, triglycerides (TG), cholesterol (CHOL), total and direct bilirubin, and blood glucose level.

It has been previously reported that 25(OH)D results obtained for patients are unaffected by multiple freeze-thaw cycles.[16] The method for quantitative determination of 25(OH)D was done using DRG® 25 (OH) vitamin D (EIA-3153) (DRG International Inc., USA). This test kit is a competitive protein binding assay for the measurement of 25-OH vit D. It is according to the competition of 25-OH vit D present in the sample with 25-OH vit D tracer, for the binding pocket of vitamin D binding protein (VDBP, Gc-globulin).

Genotyping of single-nucleotide polymorphisms

Venous blood (4 mL) was collected from all participants and stored in ethylene diamine tetra-acetic acid containing tubes at −80°C. Whole blood cell DNA was extracted by Qiagen Blood DNA mini kit (Qiagen China [Shanghai] Co, Ltd, Shanghai, China) following the manufacturer's protocol. TaqMan probe genotyping approach was employed to analyze SNP rs2228570 genotypes. TaqMan probe was designed and synthesized by Applied Biosystems (Waltham, MA; www.appliedbiosystems.com). The volume of a reaction mixture consisting of 20 ng of genomic DNA, 0.4 μL of the primers, probing mixture (single-nucleotide polymorphism TaqMan assay mix), 7.5 μL of universal polymerase chain reaction (PCR) mixture (TaqMan genotyping master mix), and water to 15 μL. PCR reactions were performed as follows: 95°C for 10 min, then 40 cycles of 95°C for 15 s, and 60°C for 1 min. Plates were read by ABI7900 real-time PCR instrument. The genotypes results were analyzed by the ABI SDS2.3 software.

Statistical analysis

The collected data were analyzed using the SPSS program version 20 (IBM Corp., Armonk, NY, USA). Difference among groups was performed using Mann-Whitney U test. The association between two variables in the same group was performed using Pearson product-moment correlation in the case of parametric values and Spearman in case of non-parametric values, whereas, median survival time was estimated by plotting Kaplan-Meier curve. The univariate logistic regression analysis was done to assess the association of different genotypes with PC risk, odds ratios, and 95% confidence intervals were also calculated. Hardy–Weinberg equilibrium test was also used to determine VDR rs2228570 genotypes distributions.

 Results



Baseline demographic characters of the PC patients and control are shown in [Table 1] and [Table 2]. The PC patients were 31 males and 16 females with mean age 62 years, ranging from 44–77 years. On comparison with the control group, they showed statistical differences regarding age (P 0.003), residency (P 0.000), smoking (P 0.008), coffee drinking (P 0.003), and high meat consumption (P 0.000), whereas we did not find any significance regarding sex, alcohol intake, and obesity.{Table 1}{Table 2}

Biochemical analysis results

[Table 2] shows the biochemical results of the PC group and the control group, the enzymatic activity of the enzymes ALT, AST, GGT, and ALP have shown statistically significant differences between the PC patients and the control group with 1.8, 3.6, 6.6, and 7.3-fold increase, respectively.

We also found a statistical difference regarding blood glucose level and lipid metabolism profile (TG and CHOL) with 2.5 and 1.3-fold increase for both TG and CHOL, also the prothrombin time expressed as an international normalized ratio was 1.3-fold longer (P < 0.001).

We found higher serum levels of vitamin D in PC patients with mean 35 ng/mL, whereas it was only 24 ng/mL for the control group with statistically significant difference between them.

Vitamin D deficiency was defined as a serum 25(OH)D level of less than 20 ng/mL; vitamin D insufficiency was defined as a 25 (OH) D level of 20 ng/mL to 30 ng/mL; and vitamin D sufficiency was defined as a 25(OH)D level of at least 30 ng/mL.[17] From the PC group, we found only 4 cases (8.5%) that showed a deficiency, 17 cases (36%) showed insufficiency, and 26 cases (55%) showed vitamin D sufficiency.

One-year survival results for the deficient and sufficient groups were 25% and 15%, respectively, whereas none of them reached 5 years survival. Results also showed statistical significance between the three studied groups P < 0.0001 using Log-rank (Mantel-Cox) test and log-rank test for trend. Median survival was 9.5 months for a deficient group and 6 m for sufficient group, whereas it was only 5 m for the insufficient group as shown in [Figure 1].{Figure 1}

Vitamin D serum level was analyzed and correlated with many parameters including age, sex, BMI, tumor stage, and diet. There were a statistically significant difference regarding age and sex, but we did not reach any significance regarding BMI, diet, and tumor stage; interestingly, the mean concentration of vitamin D serum level in early stages (I, II) is higher than late stages (III, IV) (36.7 ng/mL vs. 33.8 ng/mL).

SNP rs2228570 genotypes results

VDR Fok1 genotypic distributions in control were calculated according to the Hardy-Weinberg equilibrium, with P values 0.72 [Table 3].{Table 3}

As shown in [Table 4], VDR Fok1 polymorphism distribution in PC was CC 26 (55.3%), CT 16 (34%), and TT 5 patients (10.7%). For the control group, CC was found in 24 (64.8%), CT in 12 (32.4%), and TT genotype was found only in one individual 1 (2.8%) with no statistically significant difference between the two studied groups. We did not find any statistically significant difference when we correlated the rs2228570 polymorphisms with stage and vitamin D serum levels.{Table 4}

Although CC genotype patients reached 5-year survival, we did not find differences when comparing the different genotypes with survival as shown in [Table 5].{Table 5}

As shown in [Table 6], the univariate regression analysis did not show that any allele could increase the risk for PC, although the frequency of T allele in PC group was 5 times its frequency in the control group.{Table 6}

 Discussion



Recent studies that link vitamin D status with solid tumors have shown variable results. Although vitamin D sufficient level was associated with a better prognosis and survival in colon, melanoma, and bladder cancer patients, there was no statistically significant association in patients with non-small-cell lung cancer, whereas there were controversial results with breast cancer.[18]

To our knowledge, this is the first study carried out on Egyptian patients to examine PC in association with pre-diagnostic vitamin D status, as assessed with 25(OH)D concentrations; unexpectedly, we observed a 1.5-fold increase in serum level of vitamin D for PC when compared with non-PC subjects.

The results of reports that link the vitamin D status with PC are contradictory. Many reports have shown that inadequate vitamin D serum level increases risk to PC.[15] Many other reports have shown that dietary vitamin D or circulating concentrations of 25 hydroxy vitamin D are not associated with the risk of PC.[19] In a recent meta-analysis study by Zhang et al.,[20] they showed that there were no significant associations between vitamin D intake or serum 25(OH)D levels and PC hazards.

Many studies in different populations have reported that subjects with low 25(OH)D concentrations or in variance (<25 nmol/L) relative to higher concentrations being associated with a lower risk for prostate and esophageal cancer[21],[22] that may be explained by 25(OH)D connection to growth factors, which might impact tumor growth.[23],[24] Mechanisms that may explain these associations are unclear, and there is a lack of understanding of the molecular mechanism by which how vitamin D and its receptor can regulate the expression of genes involved in carcinogenesis.

The reports that investigated the expression of the VDR and its polymorphisms that modulate the activity of vitamin D, which may in turn affect the prognosis and survival of PC patients, are inconsistent. Some reports have shown that some VDR gene polymorphisms are associated with better prognosis and improved survival,[25] whereas others have shown that SNP in VDR did not provide any significant prognostic value.[26] Fok1 which is one of the most frequently analyzed SNP has shown contradictory results when linked with cancer risk.[27] The results of the current study showed that Fok1 polymorphism is not associated with risk of PC in Egyptian patients, which is in variance with other reports.[27] This variation in our results may be because of different ethnicity as those studies were carried on the Chinese population who have different dietary habits, different climate, and other genetic factors, which may interfere with Fok1 polymorphisms. Although it is worthy to note the number of patients in our study is a limiting factor that may interfere with our statistical results, we found that the frequency of the TT genotype in PC was 5 patients (80 %) of the total genotype in the two groups we studied which did not reach statistical significance.

Another finding in our study is a lack of association of serum 25(OH)D levels and BMI, tobacco smoking, and cancer stage, which is consistent with results obtained by other results.[28]

Results of the present study also have shown that there is a statistically significant difference between PC patients and healthy controls regarding meat consumption. Compounds such as heterocyclic amines, polycyclic aromatic hydrocarbons, and N-nitroso compounds that can be formed during cooking have been reported as possible pancreatic carcinogens.[29]

In conclusion, low serum vitamin D or VDR-SNP is not a risk factor for PC in Egyptian patients. Recommendations to increase vitamin D concentrations in healthy persons for the prevention of cancer and improving overall survival should be carefully considered. The main limitation of the study was the small number of patients. Hence, studies with a large number of patients should be carried out to confirm these results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin2015;65:5-29.
2Klapman J, Malafa MP. Early detection of pancreatic cancer: Why, who, and how to screen. Cancer Control 2008;15:280-7.
3Ibrahim AS, Khaled HM, Mikhail NN, Baraka H, Kamel H. Cancer incidence in egypt: Results of the national population-based cancer registry program. J Cancer Epidemiol 2014;2014:437971.
4Lowenfels AB, Maisonneuve P, Lankisch PG. Chronic pancreatitis and other risk factors for pancreatic cancer. Gastroenterol Clin North Am 1999;28:673-85, x.
5Larsson SC, Orsini N, Wolk A. Body mass index and pancreatic cancer risk: A meta-analysis of prospective studies. Int J Cancer 2007;120:1993-8.
6Berrington de Gonzalez A, Sweetland S, Spencer E. A meta-analysis of obesity and the risk of pancreatic cancer. Br J Cancer 2003;89:519-23.
7Bulathsinghala P, Syrigos KN, Saif MW. Role of vitamin d in the prevention of pancreatic cancer. J Nutr Metab 2010;2010:721365.
8Garland CF, Mohr SB, Gorham ED, Grant WB, Garland FC. Role of ultraviolet B irradiance and vitamin D in prevention of ovarian cancer. Am J Prev Med 2006;31:512-4.
9Stolzenberg-Solomon RZ, Jacobs EJ, Arslan AA, Qi D, Patel AV, Helzlsouer KJ, et al. Circulating 25-hydroxyvitamin D and risk of pancreatic cancer: Cohort Consortium Vitamin D Pooling Project of Rarer Cancers. Am J Epidemiol 2010;172:81-93.
10Guyton KZ, Kensler TW, Posner GH. Vitamin D and vitamin D analogs as cancer chemopreventive agents. Nutr Rev 2003;61:227-38.
11Skinner HG, Michaud DS, Giovannucci E, Willett WC, Colditz GA, Fuchs CS. Vitamin D intake and the risk for pancreatic cancer in two cohort studies. Cancer Epidemiol Biomarkers Prev 2006;15:1688-95.
12Stolzenberg-Solomon RZ, Vieth R, Azad A, Pietinen P, Taylor PR, Virtamo J, et al. A prospective nested case-control study of vitamin D status and pancreatic cancer risk in male smokers. Cancer Res 2006;66:10213-9.
13Raimondi S, Johansson H, Maisonneuve P, Gandini S. Review and meta-analysis on vitamin D receptor polymorphisms and cancer risk. Carcinogenesis 2009;30:1170-80.
14Ogunkolade BW, Boucher BJ, Prahl JM, Bustin SA, Burrin JM, Noonan K, et al. Vitamin D receptor (VDR) mRNA and VDR protein levels in relation to vitamin D status, insulin secretory capacity, and VDR genotype in Bangladeshi Asians. Diabetes 2002;51:2294-300.
15Cho M, Peddi PF, Ding K, Chen L, Thomas D, Wang J, et al. Vitamin D deficiency and prognostics among patients with pancreatic adenocarcinoma. J Transl Med 2013;11:206.
16Antoniucci DM, Black DM, Sellmeyer DE. Serum 25-hydroxyvitamin D is unaffected by multiple freeze-thaw cycles. Clin Chem 2005;51:258-61.
17Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. AmJ ClinNutr 2006;84:18-28.
18Wesa KM, Segal NH, Cronin AM, Sjoberg DD, Jacobs GN, Coleton MI, et al. Serum 25-hydroxy vitamin D and survival in advanced colorectal cancer: A retrospective analysis. Nutr Cancer 2015;67:424-30.
19Liu SL, Zhao YP, Dai MH, You L, Wen Z, Xu JW. Vitamin D status and the risk of pancreatic cancer: A meta-analysis. Chin Med J 2013;126:3356-9.
20Zhang X, Huang XZ, Chen WJ, Wu J, Chen Y, Wu CC, et al. Plasma 25-hydroxyvitamin D levels, vitamin D intake, and pancreatic cancer risk or mortality: A meta-analysis. Oncotarget 2017;8:64395-406.
21Gaksch M, Jorde R, Grimnes G, Joakimsen R, Schirmer H, Wilsgaard T, et al. Vitamin D and mortality: Individual participant data meta-analysis of standardized 25-hydroxyvitamin D in 26916 individuals from a European consortium. PloS One 2017;12:e0170791.
22Mikhak B, Hunter DJ, Spiegelman D, Platz EA, Hollis BW, Giovannucci E. Vitamin D receptor (VDR) gene polymorphisms and haplotypes, interactions with plasma 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and prostate cancer risk. Prostate 2007;67:911-23.
23Chen W, Dawsey SM, Qiao YL, Mark SD, Dong ZW, Taylor PR, et al. Prospective study of serum 25(OH)-vitamin D concentration and risk of oesophageal and gastric cancers. Br J Cancer 2007;97:123-8.
24Maestro B, Davila N, Carranza MC, Calle C. Identification of a Vitamin D response element in the human insulin receptor gene promoter. J Steroid Biochem Mol Biol 2003;84:223-30.
25Innocenti F, Owzar K, Cox NL, Evans P, Kubo M, Zembutsu H, et al. A genome-wide association study of overall survival in pancreatic cancer patients treated with gemcitabine in CALGB 80303. Clin Cancer Res 2012;18:577-84.
26Arem H, Yu K, Xiong X, Moy K, Freedman ND, Mayne ST, et al. Vitamin D metabolic pathway genes and pancreatic cancer risk. PloS One 2015;10:e0117574.
27Li L, Shang F, Zhang W, Zhang C, Li J, Wang C, et al. Role of vitamin D receptor gene polymorphisms in pancreatic cancer: A case-control study in China. Tumour Biol 2015;36:4707-14.
28Yuan C, Qian ZR, Babic A, Morales-Oyarvide V, Rubinson DA, Kraft P, et al. Prediagnostic plasma 25-hydroxyvitamin D and pancreatic cancer survival. J Clin Oncol 2016;34:2899-905.
29Sinha R, Peters U, Cross AJ, Kulldorff M, Weissfeld JL, Pinsky PF, et al. Meat, meat cooking methods and preservation, and risk for colorectal adenoma. Cancer Res 2005;65:8034-41.