MICA-129 Met/Val Variant as Possible Biomarker of Diagnosis and Prognostic of Gastro-Intestinal Tract Carcinomas

Background: The major histocompatibility complex class I-related chain A (MICA) molecules play a pivotal role in the modulation of anti-tumor immune responses. A polymorphic change from methionine (Met) to valine (Val) at amino acid position 129 of the alpha 2 heavy-chain categorize MICA alleles into strong and weak binders for the NKG2D receptor. We investigated here whether MICA-129 alleles are associated with gastro-intestinal tract (GItract) carcinomas in Tunisian affected patients as compared to healthy controls (HC). Material and methods: 181 patients affected by colorectal cancer (CRC) and 61 patients affected by gastric cancer (GC) along with 203 healthy controls (HC) were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure. Results: We found that the MICA-129 Val/Val genotype was statistically more prevalent in patients affected both by CRC and GC as compared to HC. After stratification with poor prognostic parameters, we observed that MICA129 Val/Val genotype is significantly associated with advanced tumor extension (T3-T4), lymph node metastasis (N+), and distance metastasis (M+). In both cases, the MICA-129 Val/Val genotype seems to behave as a risk genotype and a poor prognostic biomarker in our population. Conclusion: Our findings suggest a potential tumor escape possibly due to an inability to activate NK cells and/ or to stimulate specific T lymphocytes subsets particularly active in the GI-tract. Citation: Fayza A, Arij BC, Olfa B, Nesrine O, Hajer A, et al. (2017) MICA-129 Met/Val Variant as Possible Biomarker of Diagnosis and Prognostic of Gastro-Intestinal Tract Carcinomas. J Mol Biomark Diagn S2: 031. doi:10.4172/2155-9929.S2-031


Introduction
Colorectal and gastric cancers are the most common forms of malignancies often presenting with a poor prognosis and are leading cause of cancer-related death in the world [1]. In Tunisia, the incidence of these types of cancers was increasing during the period of 1999-2003 according to the register of cancer. The incidence rate is 5.4 cases per 10 5 in males, however, in females the incidence rate is 4.8 cases per 10 5 [2]. The development of these complex multifactorial malignancies is under the influence of the patient genetic background [3] and environmentally related factors [4]. Among the genetic loci that could constitute a potential link between the genetic and the environmental components of immune response, the major histocompatibility complex (MHC) class I chain-related A (MICA) gene is an attracting candidate. The MICA gene is at the centromeric end of the classical class I region approximately 46.4 Kb from HLA-B [5]. MICA (11.7 Kb) is transcribed into an mRNA of 1382 bp, giving rise to a 383-amino acid polypeptide of 43 kDa [6]. The MICA protein comprises a transmembrane MHC-I alpha-like chain and is not associated to the β-2-microglobulin and does not bind to peptides [7]. In humans, the expression of MICA is restricted to gastro-intestinal epithelium, endothelial cells and fibroblasts [7,8]. Under pathological conditions, MICA expression is induced by factors of cellular stress and can be up-regulated by viral and bacterial infections [9,10]. It is expressed in certain epithelial tumors and lymphoproliferative malignancies including multiple myeloma, lung, kidney, prostate, breast and colon [11][12][13][14]. The cognate receptor of MICA is a type II C-lectin-like protein designated as NKG2D. This receptor is present on natural killer (NK) cells, most γ/δ T cells and CD8+ α/β T cells but is absent on CD4+ α/β T cells. The engagement of MICA with NKG2D strongly activates NK cells and provides costimulatory signals to T cells, enhancing their cytolytic activity and cytokine production [15,16]. Consequently, the expression of MICA on tumor cells has been proposed to play a critical role in tumor immune surveillance. Whereas the expression of MICA induces a strong tumor antigen specific immune response, its absence, or down-regulation results in tumor escape from NK and T cell attack [17][18][19].   129 polymorphism was associated with several pathologies [21][22][23][24][25][26][27][28]. However, MICA-129 polymorphism in gastric and colorectal cancer susceptibility has not been studied so far.
In this case-control study, we sought to determine whether or not the MICA-129 polymorphism is associated with CRC and GC in the Tunisian population. Besides, we have tested the possible association of this polymorphism with clinical features.

Patients and control groups
In this study, 181 consecutive CRC patients (100 men and 81 women, age, 16-82 years) and 61 GC patients (43 men and 18 women, age, 20-89 years) were enrolled from the Institute of Cancer "Salah Azaiz" in Tunis, Tunisia. The average age of all cases at diagnosis was 57 years with a range of 16 to 89 years. Demographic and clinicopathological characteristics of the study subjects are given in Table 1. The data collected included stage, lymph node status, differentiation and histological type of tumor, besides we collected data concerning chemotherapy and surgery treatment. Some patients received FOLFOX treatment as well as FOLFIRI combination, whereas other received monotherapy treatment such as XELODA and LV5-FU2. Patients were assessed before the initiation of chemotherapy and every 2 weeks during treatment. In addition, a total of 203 age and sex-matched healthy controls were recruited in this study from "Clinical Biology Department, Salah Azaiz Institute". These controls were considered without any history of malignancy, the sex ratio of the group was 0.97 (100 men and 103 women), and the average age was 54 years with a range from 16 to 93 years [29,30]. The patients and healthy controls were not consanguineous. In our study, written informed consent was obtained from all participants before their participation and is approved by the Ethical Committee of Salah Azaiz institute.

Statistical analysis
Statistical analysis was performed using the compare V.2.1 statistical software package. Both allele and genotype frequencies of MICA-129 polymorphism were compared between cases and controls using the χ 2 test or Fisher exact test for a low number of cases. Corrected P value (Pc) was obtained by multiplying the P value by the number of alleles tested according to Bonferroni's correction. The odds ratios (OR) were calculated as estimates of relative risk for disease, and 95% confidence intervals (CI) were calculated for all observed allele frequencies. P<0.05 was considered to be statistically significant.

Characteristics of prognostic factors, surgery and chemotherapy treatment in patients with gastrointestinal cancer
A total of 181 cases with CRC and 61 cases with GC were included in this study. Stratified phenotypic characteristics of the studied cohort are summarized in Table 1. At the time of diagnosis, the vast majority of the patients with CRC presented an advanced tumor extension T3-T4 (90%) with lymph node metastasis (60%). Besides, we observed that the vast majority of the patients with GC presented only lymph node metastasis (63%).
About 123 CRC and 34 GC were followed and we have grouped some clinico-pathological information listed in Table 2, such as histological type, evolution, surgery, and chemotherapy treatment.
We observed that for CRC, the majority of the histological types were adenocarcinoma (30%) and adenocarcinoma Lieberkuhnien (32%) and were most frequently of a well differentiated histological grade (56%). As for GC cases, the majority of patients had an adenocarcinoma (32%) and adenocarcinoma Signet Ring Cell type (35%). In addition, they had low and moderate histological grade (24%, 26% respectively). Furthermore, the majority of both patients did not have a progressive disease (85% for CRC and GC). Moreover, for CRC, most of the patients have received adjuvant chemotherapy based on biotherapy treatment combination FOLFOX (5FU Oxaliplatine) and FOLFIRI (5FU Irinitecan) (50%). However, for GC, most of the patients have received adjuvant chemotherapy LV5-FU2 (53%).
Nevertheless, we did not find any association of MICA-129 Met/ Met genotype with the risk of GIC (P> 0.05).

Interaction between MICA-129 Val/Val genotype and treatment
Concerning the treatment presented in Table 5  However, in patients with CRC, we did not find any interaction between surgery, chemotherapy, radiotherapy and MICA-129 Val/Val genotype (Table 5).

Discussion
MICA is constitutively expressed within the gastro-intestinal tract and is upregulated in response to stress. It is a ligand for the NKG2D receptor on both CD8+ T cells and NK cells, reducing the threshold for lysis. Tumors may up-regulate MICA in response to physical stress such as anoxia; however, this may make them susceptible to immune attack, resulting in control of tumor growth and a more favorable prognosis.
In particular, we focused our attention on functionally relevant characteristics of MICA polymorphism. This SNP (rs1051792) of MICA gene resulting in the MICA-129 Met/Val dimorphism was the first MICA polymorphism for which a functional consequence was and categorizes that MICA-129 Met variants as high and MICA-129 Val variants as low avidity NKG2D ligands [28,29].
In our study, we shed the light for the first time on the crucial role of MICA-129 polymorphism in gastro-intestinal cancer and its association with clinical features in Tunisian population. We found that the frequency of the MICA-129 Val allele and MICA-129 Val/ Val genotype were increased in patients with gastro-intestinal cancer. These observations suggested that the MICA-129 Val is an allele dosedependent manner that increased the risk of CRC and GC, and the effect is recessive. Our findings and interpretations were in disagreement with the results published by Gong et al. where they not found an association between this dimorphism and the risk of development of colorectal cancer [24]. This discrepancy could be explained by the small size of the sample in an important genetic background like Chinese population (117 colorectal cancer patients and 113 healthy individuals) or by the   [22,26] and a number of autoimmune diseases, such as early-onset ankylosing spondylitis [21], rheumatoid arthritis [31], inflammatory bowel disease [32], systemic lupus erythematosus [33], Type 1 diabetes [34] and psoriatic disease [35]. Altogether, these data highlighted the potential role of MICA-129 in tumor and auto-immune susceptibility. The functional consequence of MICA-129 variants is investigated in many studies. In patients with ulcerative colitis, the MICA-Val/Val was associated with higher sMICA serum levels [36] as well as in patients with hepatitis B virusinduced hepatocellular carcinoma and healthy controls [25]. But, it was unclear however whether the MICA-129 dimorphism has a direct effect on the generation of sMICA and affecting MICA shedding. Recently, Isernhagen et al. clarify whether the MICA-129 not only affects NKG2D signaling but also directly affects plasma membrane expression and shedding and associated with high sMICA concentration [28].
Taken together, these results led us to hypothesize that the weaker binder MICA-129 Val allele associated with high concentration of sMICA might downregulate the activation and co-stimulate NK cells or cytotoxic T cells and thereby may allow tumor escape to the immune system and that play important roles in the development of gastro-intestinal cancer in Tunisia. However, these suggestions must be confirmed by additional functional and association studies. Therefore, after stratification with TNM, we found that MICA-129 Val/Val genotype is strongly associated with a poor prognosis in CRC (Age >50 years old), advanced Tumor extension (T3-T4), lymph node metastasis (N+) and distance metastasis (M+), moderate and well differentiated histological grade. This association with disease progression here in observed raises the possibility that the weaker binder Val allele may have an impact on the disease extension.
Furthermore, based to previous data suggesting that MICA may have an unrecognized role in cancer therapy, we investigated here the relationship between MICA dimorphism and treatment. We found that the association between MICA-129 Val/Val genotype and chemotherapy neoadjuvant treatment increased 5.67 fold the risk of gastric cancer in our population. In this context, our results could be explained by the recent study of Keisuke et al. where they demonstrate that chemotherapy may inhibit MICA expression in hepatocellular carcinoma (HCC) and suggesting that efficient activation of liver innate immunity after anti-HCC chemotherapy treatment might represent a particularly promising approach to suppress tumor growth [37]. While, this recent progress of therapy sheds light on the important implication of MICA in a good prognosis of patients and in the regression of tumor in and suggests a promising aspect for chemo-immunotherapy against human HCC [37].
A potential limitation of this study is that the number of GC subjects was relatively fewer than those in past reports on the relationships between MICA-129-Met/Val polymorphism and cancer.
However, despite this limitation, we were able to show an association between MICA-129 variant and gastric cancer in Tunisian population. Additionally, we have found that MICA-129 Val/Val genotype is strongly associated with a poor prognosis correlations and treatment in GC.
Interestingly, our preliminary results should be interpreted with caution and will require confirmation in larger populations. Moreover, to draw comprehensive and more reliable conclusions, it is necessary to integrate more studies exploring the functional data (sMICA serum levels) in correlation with our genetics results.

Conclusion
These findings indicate the relevance of MICA-129-Met/Val polymorphism (weak/strong binders of NKG2D receptor) as a good biomarker of diagnosis and prognosis of GIC in Tunisian population. If these findings are to be confirmed with a larger patient cohort, by additional functional and association studies, the novel therapeutic intervention involving MICA can be envisaged.