Original Research
2017 March
Volume : 5 Issue : 1


Association of CTLA-4 A/G polymorphism with occurrence of ocular squamous cell carcinoma

Aparna Dwibedy, Sheetal Bakshi, Jayanti Mania Pramanik, Vidyashankar B, Avinash Funde Mahadeo, Priyanka Wagh

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 Aparna Dwibedy1,*, Sheetal Bakshi2, Jayanti Mania Pramanik3, Vidyashankar B4, Avinash Funde Mahadeo5 and Priyanka Wagh6

1Department of Opthalmology, Krishna Institute of Medical Sciences, Minister Road, Secunderabad-500003, Telangana, India

2Department of Ophthalmology, Sanjeevani eye hospital, Vitthalwadi, Ulhasnagar, Maharashtra 421003

3Department of Infectious Diseases Biology, National Institute for Research in Reproductive Health (ICMR), Parel, Mumbai-400012

4 Department of Ophthalmology, K. B. Haji Bacchooali Eye and ENT hospital, Parel, Mumbai-400012

5Department of Ophthalmology, Yashodeep superspeciality netralaya, Swami samarth road, Pathardi, Ahmednagar, Maharastra 414102

6Department of Infectious Diseases Biology, NIRRH, Parel East, Mumbai, Maharashtra 400012

*Corresponding author: Dr. Aparna Dwibedy, Consultant Orbit and Oculoplasty, Department of Opthalmology, Krishna Institute of Medical Sciences, Minister Road, Secunderabad-500003, Telangana, India. Email: aparnadwibedy84@gmail.com

 

Received 09 September 2016; Revised 19 November 2016; Accepted 28 November 2016; Published 08 December 2016

 

Citation: Dwibedy A. Bakshi S, Pramanik JM, Vidyashankar B, Mahadeo AF, Wagh P. Association of CTLA-4 A/G polymorphism with occurrence of ocular squamous cell carcinoma. J Med Sci Res. 2017; 5(1):1-4. DOI: http://dx.doi.org/10.17727/JMSR.2017/5-1

 

Copyright: © 2017 Dwibedy A. Published by KIMS Foundation and Research Center. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.



Abstract

Introduction: The association of polymorphisms of genes controlling the immune regulation with development of cancers and autoimmune diseases, has been a heavily researched area with the hope of probable discovery of a biomarker or antitumour vaccine. The most commonly investigated polymorphism is CTLA-4 +49A/G. However, no research has been carried out to find out association between eye cancers and the same polymorphism till date, to the best of our knowledge.

Purpose: To find out association between CTLA-4 +49A/G polymorphism and ocular surface squamous neoplasia (OSSN).

Methods: 12 histologically proven OSSN patients were taken as subjects. 20 cataract patients were taken for comparison. This data was further compared with the published data on 103 individuals from an Indian study. DNA extraction, SNP selection and genotyping was done. Odds ratio, confidence interval and statistical significance were found out.

Results: 95% confidence interval was 0.044-1.521 between OSSN cases and cataract group (p=0.144). 95% confidence interval of the OSSN cases and healthy control group was 0.14-2.375 (p=0.544).

Conclusions: Statistically significant difference was not found in the frequency of polymorphism between OSSN cases and groups taken for comparison, in this pilot study. Larger number of OSSN cases need to be evaluated for the polymorphism.

 

Keywords: CTLA4; OSSN; +49 A/G Polymorphism; PCR-RLFP; Immune regulation

Full Text

 

Introduction

Ocular surface squamous neoplasia (OSSN) is the third most common conjunctival malignancy Worldwide. It is most common in tropical regions of the world. It has an incidence of 37.3per 106 amongst all eye cancers and 8.4 per 106 amongst squamous cell carcinomas of eye [1]. Several factors have been implicated in development of OSSN. It may arise from dysfunctional limbal stem cells having been altered by various mutagenic agents such as ultraviolet (UV) radiation, infection with human papillomavirus (HPV) etc. [2]. Light pigmentation of hair and skin, vitamin A deficiency, heavy smoking, ocular injury are other risk factors. However, majority of susceptible peopledo not develope OSSN. Host genetic factors must also play a role in persistence of mutagenic effects of UV radiation, HPV and human immunodeficiency virus (HIV) infection etc., in subsequent development of OSSN. The genetic factors haven’t received nearly the same amount ofresearch attention as the mutagenic agents.

 

The human body generates an immune response to tumours, but it is generally ineffective at causing tumour destruction. One possible reason is that T-cell activation will not occur until two separate signals are received by the cell. The first signal is specific antigen recognition, which requires T-cell receptors to recognise and bind to major histocompatiblity complex (MHC) molecules of antigen presenting cells (APC). The second signal is Ag independent, generated by interaction between CD28 on the T-cell surface and its ligands CD80 and CD86 on APCs [4-7].

 

CTLA-4, a CD28 homologue is a glycoprotein expressed on activated T-cells and its function is to down regulate T-cell response [8-10]. CTLA-4 blockade leads to enhancement of immune response [11], rejection of tumours [12] or even cure of tumours in mice when used incombination with antitumour vaccines [13]. One of the most frequently studied polymorphisms is A/G transition in exon 1 at position +49 [14]. Results of studies suggest that a G-allele instead of an A allele at position +49 can attenuate the CTLA-4 driven down-regulation of T-cell response [15-17].

There have been three meta-analysis which have tried to assimilate all studies on association of CTLA-4 polymorphisms with various cancers. None of the studies include any eye cancer. Hence, the present study was undertaken to evaluate if there is any association between CTLA-4 polymorphism with OSSN.

 

Materials and methods

Study subjects: This was a hospital based study taking histologically proven OSSN patients and patients with cataract, for comparison. The OSSN cases and cases with cataract were age-matched. Data for comparison from a published report of one of the authors on a group of healthy individuals, who didn’t suffer from any co-morbidities or cancers was included for further comparison [18]. All patients (OSSN and cases with cataract) were provided written informed consent. Ethical clearance was obtained from both the institutes.

 

DNA extraction, SNP selection and genotyping

Blood samples (2ml) were collected by using EDTA vacutainers. The QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany) was used to isolate genomic DNA from peripheral blood lymphocytes and DNA samples were frozen at -80oC. Genotypes of CTLA4 +49G>A site were analysed by using polymerase chain reaction–restriction fragment length polymorphisms (PCR-RLFP) method (Figure 1) [18]. The association of CTLA4 +49A/G genotypes with the risk 45 of OSSN was evaluated by odds ratio (OR) and corresponding 95% confidence intervals (CIs). An internet based Hardy-Weinberg equilibrium (HWE) calculator was used to measure the deviation from the HWE among the controls.

 

Statistical analysis was implemented using SPSS version 16.0 and Epi-info version 6.0. Statistical significance was defined as P<0.05 with two tailed for all statistical analyses.

Results

There were 12 histologically proven OSSN patients and 20 patients with cataract. Of the 12 histologically proven cases, 9 were intraepithelial OSSN (Figure 2) and 3 were invasive OSSN. Mean age of the OSSN patients and cataract patients was 54.1 years. Among the OSSN patients 8 were males and 4 were females. Among the cataract patients 14 were males and 6 were females. Published data available from 103 healthy individuals, who didn’t suffer from any eye problems, co-morbidities or cancers were taken for comparison. Among them 58 were males and 42 were females. All were of western Indian ethnicity.

A/G polymorphism was seen in 4 out of 12 cases of OSSN, in 13 out of 20 cases of cataract, while this was reported in 47 of 103 healthy individuals. The 95% confidence interval was 0.044-1.521, which suggests no difference in the frequency of the polymorphism between case and cataract group (Table 1). The 95% confidence interval of the cases and healthy group was 0.14-2.375 which again suggests no difference in the frequency of the polymorphism (Table 2). Because of small number of samples available for each tumour grade, we could not get any statistically significant correlation with +49A/G polymorphism.

 

Table 1: Comparision of CTLA4 +49A/G polymorphism in OSSN cases and cataract patients.

CTLA-4

(+ 49A/G)

OSSN cases

N=12

Percentage

Cataract cases

N=20

 

Percentage

OR

P-value

95% CI

A/A

4

33.33

5

25

1.5

0.696

0.241-9.429

A/G

4

33.33

13

65

0.269

0.144

0.044-1.521

G/G

4

33.33

2

10

4.5

0.165

0.526-45.985

Allele

 

 

 

 

 

A

12

50

23

57.5

G

12

50

17

42.5

 

 

0.2482(HW)

 

0.1401(HW)

 

 

 

Table 2: Comparision of CTLA4 +49A/G polymorphism in OSSN cases and healthy individuals.

CTLA-4

(+ 49A/G)

Cases

N=12

Percentage

Healthy individuals [Ref.18]

N=103

Percentage

OR

P-value

95% CI

A/A

4

33.33

26

25.24

1.481

0.508

0.34-6.078

A/G

4

33.33

47

45.63

0.596

0.544

0.14-2.375

G/G

4

33.33

30

29.12

1.217

0.747

0.282-4.943

Allele

 

 

 

 

 

A

12

50

99

48.06

G

12

50

107

51.94

 

 

0.2482

(HW)

 

0.3828

(HW)

 

Discussion

In 2010, Zheng J et al. [19] found out in their metanalysis that presence of CTLA4 +49A/G was associated with increased risk of developing solid tumours (lungs, breast, colorectal, skin, nasopharyngeal, cervical, esophageal and oral squamous cell  carcinoma) but not non-solid tumors. They also found that the risk was higher in Asians and Caucasians. Gao X et al. [20] published in their metanalysis that +49A/G polymorphism was associated with decreased risk of cancer amongst Asians but not among Europeans. However no eye cancer were included in either of the metanalysis. Publication bias could be a reason why studies with negative result have been missed out of metanalyses.

 

Acknowledgements

Krishna Institute of Medical Sciences (KIMS), Secunderabad-500003; Kb Haji Bachooali Ophthalmic & Ent Hospital, J.M. Street, Parel, Mumbai-400012; and National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai-400012, Indial.

 

Conflicts of interest

Authors declare no conflicts of interest.

 

Conclusion

In this pilot study, we investigated a specific SNP (+49 A/G) of the CTLA 4 gene in patients suffering from OSSN. We did not find any statistically significant difference between the presence of +49A/G polymorphism in OSSN patients, cataract patients and normal healthy individuals. Small sample size is limitation of the study. Further studies to determine the association of +49A/G polymorphism in OSSN in large number of cases are required to confirm the contribution of this polymorphism.

 

References

 [1] Kao AA, Galor A, Karp CL, Abdelaziz A, Feuer WJ, et al. Clinicopathologic correlation of ocular surface squamous neoplasms at Bascom Palmer Eye Institute: 2001 to 2010. Ophthalmology. 2012; 119(9):1773-1776.

[2] Armstrong BK, Kricker A. The epidemiology of UV induced skin cancer. J Photochem Photobiol B. 2001; 63(1-3):8-18.

 [3] Mittal R, Rath S, Vemuganti GK. Ocular surface squamous neoplasia - Review of etio-pathogenesis and an update onclinico-pathological diagnosis. Saudi J Ophthalmol. 2013; 27(3):177-186.

[4] Azuma M , Ito D, Yagita H, Okumura K, Phillips JH, et al. B70 antigen is a second ligand for CTLA-4 and CD28. Nature. 1993; 366(6450):76-79.

[5] Freeman GJ, Borriello F, Hodes RJ, Reiser H, Hathcock KS, et al. Uncovering of functional alternative CTLA-4 counter-receptor in B7-deficientmice. Science. 1993; 262(5135):907-909.

[6] Hathcock KS, Laszlo G, Dickler HB, Bradshaw J, Linsley P, et al. Identification of an alternative CTLA-4 ligand costimulatory for T cell activation. Science. 1993; 262(5135):905-907.

 [7] Townsend SE, Allison JP. Tumor rejection after direct costimulation of CD8+ T cells by B7-transfected melanoma cells. Science. 1993 Jan 15;259(5093):368-370.

 [8] Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol. 2002; 3(11):1097-1101.

 [9] Manzotti CN, Tipping H, Perry LC, Mead KI, Blair PJ, et al. Inhibition of human T cell proliferation by CTLA-4 utilizes CD80 and requires CD25+regulatory T cells. Eur J Immunol. 2002; 32(10):2888-2896.

 [10] Schneider H, Valk E, Leung R, Rudd CE. CTLA-4 activation of phosphatidylinositol 3-kinase (PI 3-K) and protein kinase B (PKB/AKT) sustains T-cell anergy without cell death. 2008; 3(12):e3842.

[11] Allison JP, Hurwitz AA, Leach DR. Manipulation of costimulatory signals to enhance antitumor T-cell responses. Curr Opin Immunol. 1995; 7(5):682-686.

[12] Hurwitz AA, Foster BA, Kwon ED, Truong T, Choi EM, et al. Combination immunotherapy of primary prostate cancer in a transgenic mouse model using CTLA-4 blockade. Cancer Res. 2000; 60(9):2444-2448.

[13] van Elsas A, Hurwitz AA, Allison JP. Combination immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte associated antigen 4 (CTLA-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF)-producing vaccines induces rejection of subcutaneous and metastatic tumors accompanied by autoimmune depigmentation. J Exp Med. 1999; 190(3):355-366.

[14] Ueda H, Howson JM, Esposito L, Heward J, Snook H, et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature. 2003; 423 (6939):506-511.

[15] Sun T, Zhou Y, Yang M, Hu Z, Tan W, et al. Functional genetic variations in cytotoxic T-lymphocyte antigen 4 and susceptibility to multiple types of cancer. Cancer Res. 2008; 68(17):7025-7034.

[16] Kouki T, Sawai Y, Gardine CA, Fisfalen ME, Alegre ML, et al. CTLA-4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA-4 and contributes to the pathogenesis of Graves' disease. J Immunol. 2000; 165(11):6606-6611.

[17] Mäurer M, Loserth S, Kolb-Mäurer A, Ponath A, Wiese S, et al. A polymorphism in the human cytotoxic T-lymphocyte antigen 4 (CTLA4) gene (exon 1 +49) alters T-cell activation. Immunogenetics. 2002; 54(1):1-8.

[18] Gokhale P, Kerkar S, Tongaonkar H, Salvi V, Mania-Pramanik J. CTLA-4 gene polymorphism at position +49 A>G in exon 1: a risk factor for cervical cancer in Indian women. Cancer Genet. 2013; 206(5):154-161.

[19] Zheng J, Yu X, Jiang L, Xiao M, Bai B, et al. Association between the Cytotoxic T-lymphocyte antigen 4 +49G > A polymorphism and cancer risk: A meta-analysis. BMC Cancer. 2010; 10:522.

[20] Gao X, Zhang S, Qiao X, Yao Y, Wang L, et al. Association of cytotoxic T lymphocyte antigen-4 +49A/G polymorphism and cancer risk: An updated meta-analysis. Cancer Biomark. 2014; 14(4):287-294. 

 

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