The effect of LTA gene polymorphisms on cancer risk: an updated systematic review and meta- analysis

Abstract Purpose: To provide a comprehensive account of the association of five Lymphotoxin-α (LTA) gene polymorphisms (rs1041981, rs2229094, rs2239704, rs746868, rs909253) with susceptibility to cancer. Methods: A literature search for eligible candidate gene studies published before 28 February 2020 was conducted in the PubMed, Medline, Google Scholar and Web of Science. The following combinations of main keywords were used: (LTA OR Lymphotoxin alpha OR TNF-β OR tumor necrosis factor-beta) AND (polymorphism OR mutation OR variation OR SNP OR genotype) AND (cancer OR tumor OR neoplasm OR malignancy OR carcinoma OR adenocarcinoma). Potential sources of heterogeneity were sought out via subgroup and sensitivity analysis, and publication bias were estimated. Results: Overall, a total of 24 articles with 24577 cases and 33351 controls for five polymorphisms of LTA gene were enrolled. We identified that rs2239704 was associated with a reduced risk of cancer. While for other polymorphisms, the results showed no significant association with cancer risk. In the stratified analysis of rs1041981, we found that Asians might have less susceptibility to cancer. At the same time, we found that rs2239704 was negatively correlated with non-Hodgkin lymphoma (NHL). While, for rs909253, an increased risk of cancer for Caucasians and HCC susceptibility were uncovered in the stratified analysis of by ethnicity and cancer type. Conclusion: LTA rs2239704 polymorphism is inversely associated with the risk of cancer. LTA rs1041981 polymorphism is negatively associated with cancer risk in Asia. While, LTA rs909253 polymorphism is a risk factor for HCC in Caucasian population.


Introduction
Increasing studies have demonstrated that a number of proinflammatory cytokines could be associated with the development of cancer [1,2]. Lymphotoxin-α (LTA) is the predominant member of the tumor necrosis factor (TNF) ligand family, which responds to immune and inflammatory reaction and plays an important role in the pathogenesis of cancer [3,4]. The human LTA gene is located on the short arm of chromosome 6 (6p21.3) [5]. The presence of single nucleic polymorphism (SNP) may affect cytokine expression level, which might be an important mediator of cancer [6,7]. SNP rs1041981 is a mutation of LTA gene at the 804 (C/A) position of exon 3 in codon 26, causing the amino acid threonine to be asparagine, which may be related to the transcriptional regulation of LTA, then activate the lymphocytes and induce apoptosis [8]. While, SNP rs909253 is a mutation of LTA gene at 252 (A/G) position in intron 1, which may lead to increase in the transcriptional activities of LTA [1]. In addition, SNP rs2239704, rs746868 and rs2229094 are associated with the LTA expression, which may affect subsequent inflammatory responses and immunomodulatory diseases, including cancers [9,10].
There are ample evidences that have demonstrated the association between LTA polymorphisms and cancer . However, these results are inconsistent and even contradictory, which might be due to the heterogeneity within cancer types, ethnicities, source of control, Hardy-Weinberg equilibrium (HWE), The pooled results based on six included studies [11][12][13][14][15][16] (including 6427 cases and 9714 controls) indicated that no significant association between rs1041981 polymorphism and cancer risk was found. However, in the stratification analysis by ethnicity, we observed that Asian group was significantly related to a reduced risk of cancer in allelic contrast (B vs A: OR = 0.79, 95% confidence interval (CI) = 0.64-0.97, P=0.027, Figure 2) and dominant model (BB+AB vs AA: OR = 0.67, 95% CI = 0.52-0.87, P=0.002). Moreover, when the subgroup analysis was performed based on source of controls, hospital-based control group was significantly related to a decreased risk of cancer in allelic contrast (B vs A: OR = 0.69, 95% CI = 0.55-0.87, P=0.002) and dominant model (BB+AB vs AA: OR = 0.58, 95% CI = 0.42-0.78, P=0.000) ( Table 3).

rs2229094
The pooled results based on four included studies [11,[17][18][19] (including 6227 cases and 8562 controls) indicated that no significant association between rs2229094 polymorphism and cancer risk was found. Further subgroup analysis by ethnicity also indicated that no significant result was uncovered (Supplementary Table S1).

rs746868
The pooled results based on four included studies [18,[25][26][27] (including 1351 cases and 2145 controls) indicated that no significant association between rs746868 polymorphism and risk of cancer was uncovered. Moreover, in the subgroup analysis by cancer type, ethnicity and source of control, similar results were found. (Supplementary Table  S2).

Sensitivity analysis and publication bias
Sensitivity analysis were performed to evaluate the influence of each separate case-control study. The results showed that there was no material alteration in corresponding pooled ORs for rs1041981, rs2229094, rs2239704, rs746868, rs909253 (Supplementary Figures S1-S5). In addition, Begg's test and Egger's regression test were performed to evaluate the publication bias. As for rs1041981, rs2229094, rs2239704, rs746868 and rs909253, no evidence of publication bias was identified (Supplementary Table S3).

TSA
To evaluate random errors, we performed TSA ( Figure 5). This analysis showed that the cumulative z-curve did not cross the trial sequential monitoring boundary and the required information size, suggesting that more evidences are needed to verify the conclusions.

Discussion
In the present study, a total of 24 articles including 43 case-control studies were enrolled to validate the association between five LTA gene polymorphisms (rs1041981, rs2229094, rs2239704, rs746868, rs909253) and the risk of cancer. We identified that rs2239704 was inversely associated with the risk of cancer under different genetic models. However, for LTA rs1041981, rs2229094, rs746868, rs909253 polymorphisms, no significant association with cancer risk was uncovered.
In subgroup meta-analysis stratified by cancer type, we found that rs2239704 was significantly reduced NHL susceptibility. Huang et al. reported rs2239704 polymorphism was correlated with cancer and positive association in North Americans [35]. However, they included studies that contained buccal samples for SNP analysis or insufficient data studies [37][38][39]. We strictly follow the inclusion and exclusion criteria to include the literature. And our results indicated that rs2239704 was significantly reduced cancer susceptibility in mixed ethnicity, hospital-based control and polymerase chain reaction (PCR) genotyping subgroups. Despite of several possible bias, we still could conclude that rs2239704 could reduce cancer susceptibility.
In the stratified analysis of rs1041981, we found that Asians might have less susceptibility to cancer. Unlike the study by Huang et al. [35], we excluded two studies, one of which was autopsy specimen for SNP analysis [10] and the other was a study of HIV-infected patients [40]. The literature thus incorporated has a better baseline consistency and is more reflective of the real situation. Our results were consistent with the results of Huang et al. [35]. Due to the small sample size, we were unable to evaluate the role of rs1041981 in Caucasians. Larger sample size studies are needed for further evaluation. However, based on the current studies, we might conclude that rs1041981 could reduce cancer susceptibility in Asians.
For LTA rs2229094 and rs746848, only four studies reported their relationship with cancer in each group. No significant results were found. Huang et al. reported positive association between rs2229094 and cancer risk [35], which could be the bias from report by Takei et al. [10]. Because of the small sample size, we could not draw any conclusions based on current literature.
Although the overall analysis of rs909253 indicated a null result for cancer risk, the risk of cancer for Caucasians and HCC susceptibility were significantly increased in the stratified analysis by ethnicity and cancer types. In addition, some of the control groups did not match HWE, we can not exclude the possibility that may cause the bias. Then, subgroup analysis by HWE showed that HWE status did not cause the bias of results. Huang et al. did not report the relationship of rs909253 and cancer risk, because it might be present in high linkage disequilibrium with other four SNPs [8,9]. However, our results identified that the function of rs909253 was opposite to rs2239704 and rs1041981. So, further studies with larger sample size are required to identify the role of LTA rs909253 and the linkage disequilibrium with other SNPs.
In the present study, we have put great effort on carefully searching for eligible studies. In order to obtain more accurate and reliable results, we conducted a comprehensive search to verify more eligible studies. Then, we used NOS to evaluate the quality of the included studies, eliminate low-quality studies and improve overall research quality. In order to provide the sources of heterogeneity, subgroup analysis was performed by ethnicity, cancer type, source of controls, genotyping and so on. In addition, sensitivity analysis was used to confirm the stability of the studies. Egger's and Begg's tests were used to assess publication bias. However, several limitations in our study should be noted. First, small sample size limits the reliability of the results for some polymorphisms. Second, we just included the studies published in English, which may influence the effects of the polymorphisms. Third, we mainly evaluated the relationship between LTA polymorphisms with various cancers, and we could not get enough data for some cancer types. Fourth, we did not assess the linkage disequilibrium, which might not reflect the real function correctly. In future, more well-designed case-control studies are needed to investigate the functions of LTA polymorphisms.

Conclusion
Our meta-analysis suggests that LTA rs2239704 polymorphism is inversely associated with the risk of cancer, as is LTA rs1041981 polymorphism in Asia. While, LTA rs909253 polymorphism is a risk factor for HCC in Caucasians.
Further studies with larger sample size are needed to confirm these findings.