Cancer is the second most common cause of mortality globally, which led to 9.6 million deaths in 2018[1,2]. As the mean age of the global population is increasing, cancer incidence rates have also increased in most countries[3,4]. It is widely accepted that earlier detection of malignancy reduces overall mortality rates. Therefore, early monitoring of diagnostic biomarkers can help to identify individuals at risk early allowing the institution of necessary precautions.
Gene promoter methylation is known to be an important epigenetic factor that results in the deregulation of gene expression. Gene products that are crucial for the cell cycle and their related regulation are under spatiotemporal control.
One of the main phenomena contributing to the dysregulation of these important genes is hyper- or hypo-methylation, which triggers cell cycle aberration[7,8]. The gradual accumulation of methylation errors and/or mutations contributes to the initiation and progression of sporadic malignant neoplasms. Newly formed tumors usually remained undetected up to their advanced stages which dramatically reduces the possibility and chances of effective treatments. There is an inverse relation between the stage of cancer diagnosis, treatment outcome, and survival rate. Therefore early-stage cancer screening modalities have been gaining vital importance.
To date, there are a vast variety of biomarkers available[10,11,12,13] and these have been subject to intensive research in order to develop early screening tools for cancer, however, due to the conflicting data, it would be difficult to find out the suitability of any marker for early-stage cancer screening. In a review conducted by Laugsand et al. several single markers for colorectal cancer (CRC) with a sensitivity of 75% and specificity of ≥ 90% were identified. Amongst the markers listed, the ITGA4 gene has been reported to be one of the superior single markers with a specificity of 90% or more. ITGA4 gene methylation status in chronic lymphocytic leukemia (CLL) was examined by Attia et al. who concluded that hypermethylation at three different CpG islands of the ITGA4 gene is a common event in CLL when compared with healthy controls. Methylation of the ITGA4 gene emerged as having a significant negative association with the overall survival rate in pancreatic adenocarcinoma patients and was positively associated with tumor recurrence.
The ITGA4 protein is a component of integrins, α4β1 and α4β7, and has been subjected to detailed studies to date. Integrins are heterodimeric transmembrane receptors that mediate adhesion-dependent cellular functions. Various combinations of two subunits (α and β) make 24 different integrin molecules in mammalian cells. These molecules link with a variety of biological molecules and promote multiple processes such as signaling pathways, growth, inflammation, and cell migration (Fig. 1)[18,19].
Cell-to-cell adhesion is due to the family of ITGA4 integrins which are particularly important for immune function. The α4 peptide (CD49d) binds to the β1 chain (CD29) or the β7 chain producing integrins of α4β1 (VLA‐4 or very late antigen‐4) and α4β7 (molecule of lymphocyte Peyer’s patch adhesion). Alpha-4 integrins play a role in cases such as myogenesis, hematopoiesis, heart, and placental development. The alpha 4 integrins also play a role in cardiovascular disease surveillance, inflammation, and pathogenesis. α4β1 binds to vascular cell adhesion molecule‐1 (VCAM‐1) on the surface of endothelial and stromal cells and to fibronectin that is in the extracellular matrix. α4β7, on the other hand, binds to mucosal vascular addressing cell adhesion molecule‐1 (MAd‐CAM‐1). The US Food and Drug Administration (FDA) has approved natalizumab as a humanized monoclonal antibody targeting ITGA4 that treats multiple sclerosis and Crohn's disease. However, the role played by ITGA4 in tumorigenesis still remains controversial. Some studies reported that ITGA4 is a tumor suppressor, whereas a correlation of its expression with the extent of malignant cell transformation and metastasis has been proposed by others[21,22,23]. For example, a study that examined abnormally expressed integrin subunits as biomarkers in skin cutaneous melanoma revealed that ITGA4 expression levels were closely related to metastasis and pathological stage. Also, low expression of ITGA4 in CRC cells was associated with poor prognosis and results from analysis of TCGA high-throughput RNA sequencing data revealed that ITGA4 might participate in apoptosis and survival via the PI3K/Akt pathway. In spite of several recent studies, the power and suitability of ITGA4 promoter methylation for malignant tumor screening still remain inconclusive[26,27,28,29,30]. Therefore, we have conducted a systematic review and meta-analysis of the available literature to assess the significance of ITGA4 promoter methylation evaluation in CRC and other cancers screening.
Methods and search strategy
Studies were identified by the PRISMA statement. In this meta-analysis, we performed a comprehensive search related to ITGA4 gene methylation. Two researchers (S. Jafarpour and N. Vatandoost) separately searched the electronic databases: PubMed, Embase, Web of Science, Cochrane Library, and Scopus. Eligible literature was retrieved until Jan 2022 using the following terms: (“Cancer” OR “Tumor” OR “Neoplasm” OR "Carcinoma ") AND (“ITGA4” OR “integrin alpha 4” OR “antigen CD49D” OR "alpha 4 subunits of VLA-4 receptor ") AND ("methyl*" OR "epigene*"). The abstract and the title of the publications were retrieved and the full text of relevant articles was reviewed to make sure that the data of interest were included. Any conflicts were solved through discussion with a third researcher (R. Salehi).
Inclusion and exclusion criteria
The search strategy was according to PICO characteristics based on the following criteria: 1) English as the publication language; 2) original studies evaluated the association between ITGA4 methylation and any type of cancer or tumor; 3) studies using a case-control design; 4) comparison with healthy persons, or adjacent non-cancer tissues must be considered as control samples in the studies; 5) studies with sufficient data to be able to calculate the odds ratio or the number of true positives, false positives, true negatives, and false negatives. All, non-human experiments, cell line studies, non-serum/tissue/urine samples in the study, and studies with incomplete data were excluded. Other types of studies such as reviews, letters, meeting abstracts, and redundant studies were also excluded.
Data extraction and quality assessment
Two investigators (S. Jafarpour and R. Salehi) independently assessed the quality of eligible studies according to the quality assessment of diagnostic accuracy studies (QUADAS) tool in which a study with a score ≥ 9 was considered high quality. Information extracted from the studies included the first author's name, year, ethnicity, detection methods, sample size, tumor type, and experimental results. Also, additional data such as information about subgroups, primer sequences, product size, and annealing temperature were collected. Any conflicts were solved through consensus and a third researcher.
For the diagnostic meta-analysis, we extracted true positive, false positive, true negative, and false negative from each eligible study. The ORs and 95% CIs were extracted or calculated to evaluate the strength of the association between ITGA4 methylation and cancer risk. The pooled odds ratio was computed using the method of Peto because of the sparseness of contingency tables[32,33]. Synthesizing odds ratios were done using random effects meta-analysis. Heterogeneity across the enrolled studies was evaluated by Cochran's Q-statistic and I2 statistic was used to estimate the heterogeneity of the studies in the meta-analysis. Subgroup analyses and sensitivity analyses were also performed to explore the source of heterogeneity. To further assess the diagnostic performance of ITGA4 methylation, we calculated pooled sensitivity and specificity and we also drew the summary receiver operator characteristic (SROC) curve. Deeks’ funnel plot asymmetry test was performed to assess the potential publication bias. A significant probability level was considered P < 0.05. Meta-analysis was performed using the metaphor package in R 3.5 and Meta-Disc 1.4.
In the primary search based on the terms, 127 pieces of literature were identified from electronic databases (PubMed, Embase, Web of Science, Cochrane Library, and Scopus) and manual search. All studies were exported into EndNote software and two investigators (S. Jafarpour and N. Vatandoost) independently selected the studies. Subsequently, 58 studies remained after the removal of duplicates and irrelevant titles and/or abstracts. By reviewing the full text of the articles, a total of 13 studies met the inclusion criteria for the current meta-analysis, and others were removed for the following reasons: studies with insufficient data, reviews, non-human studies, and studies unrelated to this meta-analysis. Any conflicts were solved through discussion with a third researcher (R. Salehi). Fig 2 shows the flow of the chart of the study selection process.
Study characteristics and quality assessment
In the current meta-analysis, a total of 1232 tumor samples and 649 non-tumor samples were analyzed. The included studies reported about six types of cancers, including CRC (n = 6), gastric cancer (n = 2), breast cancer (n = 1), cholangiocarcinoma (n = 1), prostate cancer (n = 1), oropharyngeal squamous cell carcinoma (n = 1), and bladder cancer (n = 1). Methylation-specific polymerase chain reaction (MSP) and quantitative methylation-specific polymerase chain reaction (QMSP) were used to measure the ITGA4 methylation status in all the included studies. Samples in the studies included stool, plasma, and tissue.
About the ethnicity of the patients; 8 Asian studies, 2 Caucasians studies, and 3 mixed populations studies. More details of the studies are shown in Table 1. Based on the QUADAS assessment, all included studies met the quality requirements based on the guideline (score ≥ 9).
Meta-analysis of ITGA4 methylation in all types of tumors
ITGA4 promoter methylation was assessed among a total of 1232 cases and 649 control samples from 13 studies; two of these studies[29,38] analyzed both tissue and stool samples of the patients which are the reasons for considering the analysis of tissue and stool samples separately. Further analysis indicated high heterogeneity in the current meta-analysis (I2 = 80.1%). Therefore, a random-effect model was applied for the current meta-analysis. Our results showed a significant association of ITGA4 methylation with cancer (OR = 13.32, 95%, CI = 7.96-22.29, I2=80.1; Fig. 3). The SROC curve indicated that ITGA4 methylation might be a promising biomarker for tumor diagnosis (AUC = 0.94, Fig. 4A; pooled sensitivity = 0.95, 95%, CI = 0.94-0.97, pooled specificity = 0.57, 95% CI = 0.54-0.60, Fig. 4B).
To investigate some sources of heterogeneity, we performed a subgroup analysis according to ethnicity, tumor type, detection method, and sample type (Table 2). Comprising 4 studies, QMSP (OR = 34.26, 95%, CI = 26.25-46.49, I2 = 0.0%) resulted in a higher diagnostic value and higher sensitivity and specificity compared with the MSP method (OR = 9.77, 95% CI = 5.51-17.32, I2 = 69.7%). The diagnostic value of ITGA4 methylation as a biomarker for cancer was not substantially different between Caucasian and Asian ethnicity. Overall, the assessment of studies used tissue samples (OR = 16.66, 95%, CI = 9.77-28.41, I2 = 74.9%) for ITGA4 methylation showed a higher diagnostic value compared to other kinds of samples (OR = 7.22, 95%, CI = 2.16-24.16, I2 = 83.3%) along with a higher sensitivity (0.96 vs 0.94) and specificity (0.60 vs 0.50).
Diagnostic value of ITGA4 for colorectal tumors
Our meta-analysis showed that ITGA4 methylation could be used as a diagnostic biomarker for CRC (OR = 20.77, 95% CI = 9.15-47.15, I2 = 87.8%; Fig. 4). Subsequently, we estimated the diagnostic values of ITGA4 methylation in CRC tissues and other types of samples (stool, urine, and plasma), respectively. There was a higher pooled odds ratio using ITGA4 methylation as a diagnostic biomarker for CRC in tissue (OR = 39.49, 95% CI = 28.83-55.33, I2 = 0.0%) compared to other types of samples (OR = 8.83, 95% CI = 2.00-39.08, I2 = 88.5%; Fig. 5).
Figure 6 shows a further assessment of the diagnostic value of ITGA4 methylation in tissue samples. There was a pooled sensitivity of 0.99 (95% CI = 0.97-1.00; Fig. 5B), pooled specificity of 0.90 (95% CI = 0.86-0.93; Fig. 6C), and an AUC of 0.94 (Fig. 6)A when CRC tissue sample was used for ITGA4 methylation assessment.
Publication bias and sensitivity analysis
The funnel plot indicated that there was no publication bias in our meta-analysis (Fig. 7). The rank correlation test (Kendall's tau = -0.11, P = 0.68) and regression test for funnel plot asymmetry (z = -1.41, P = 0.157) did not infer to publication bias, too. We perform a sensitivity analysis by deleting each study one time and recomputing pooled odds ratio. Results did not differ significantly in pooled effect and heterogeneity.
The present meta-analysis was conducted to clarify the relationship between ITGA4 promoter methylation with CRC and other cancers. Thirteen studies consisting of data from a total of 1232 tumor samples and 649 non-tumor control samples were systematically analyzed. The main outcome of the study is indicative of ITGA4 methylation which is more frequently detected in tumor samples than that of normal controls. Also, the AUC for the SROC curve was 0.94 indicating that ITGA4 methylation could be considered a single biomarker for tumor diagnosis.
In order to reduce heterogeneity, sub-group and meta-regression analyses were conducted according to the tumor types, detection methods, ethnicity, and type of samples. We performed a stratified analysis based on the method used for methylation detection (Table 2). AUC and sensitivity were well comparable but specificity emerged differently between MSP and QMSP methods (Table 2). Methylation assessment methods are typically chosen based on the study design as well as the technology available in the research facility. However, in the present study based on our stratified analyses results, we decided to include papers that adopted the MSP method as well as the QMSP method.
Subsequently, subgroup analysis according to ethnicity revealed that the diagnostic value of ITGA4 methylation was not substantially different between Caucasian and Asian ethnicity suggesting that the methylation status of ITGA4 in cancer could be ethnically independent. We also analyzed ITGA4 methylation status in all types of samples (stool, urine and plasma, and tissues) and concluded that the predictive value of ITGA4 methylation in tissue samples was higher than in other kinds of samples.
Besides DNA mutations and genetic changes, epigenetic alterations are involved in cancer initiation and tumorigenesis. Chemical modifications of DNA, chromatin remodeling, and histone modifications are common epigenetic events involved in the pathogenesis of human cancers[41,42]. Among these epigenetic modifications, DNA methylation has a prominent role and is considered an early signature of malignant tumors.
Given the multiple functions of integrins in malignant transformation and metastasis, it is clear that different results have been reported regarding the ITGA4 methylation status in malignancies[19,44]. High levels of ITGA4 play a dual role in cancer, preventing cancer cells from separating and invading, and facilitating metastasis of cancer cells by binding to surface ligands in endothelial cells. ITGA4 expression is found in a subset of breast cancer. Hypermethylation of ITGA4 is strongly associated with HER2-positive tumors. The presence of α4 in ITGA4 appears to be not beneficial for HER2-positive tumors, although its signaling pathways and molecular mechanism are not fully investigated. ITGA4 gene methylation status and gene expression pattern in CLL was examined by Attia et al. who stated that the ITGA4 expression pattern appears to be regulated at the mRNA level by local methylation CpG sites. Methylation of ITGA4 in CpG sites-2 and 3 was detected in CLL patients with del13q14+ and hypermethylation of ITGA4 in CpG sites-1 can be a potential prognostic biomarker for CLL patients. Aberrant promoter methylation of the ITGA4 gene was reported in different malignancies including CRC[46,47], cholangiocarcinoma, breast, and gastric tumors. To the best of our knowledge, this is the first diagnostic meta-analysis to evaluate the diagnostic value of ITGA4 methylation for CRC and other malignancies and our result here showed that ITGA4 could be effective for the diagnosis of cancer.
A considerable number of included articles in our meta-analysis were related to colorectal cancer. It seems that the methylation of ITGA4 is more widespread in CRC than in other cancers. Therefore, we performed a similar analysis to evaluate the diagnostic values of ITGA4 methylation in CRC patients. The outcomes indicated that ITGA4 methylation may be a more reliable diagnostic biomarker for CRC than other cancers. Pulkka et al. showed that ITGA4 is the main integrin in the pathogenesis of gastrointestinal stromal tumors and is associated with poor overall survival. Our results may support a model that DNA methylation mediates downregulation and are consistent with the study carried out by Mo et al., which revealed that downregulation of ITGA4 was associated with poor prognosis in CRC patients and ITGA4 could be an early predictor of CRC.
The gene methylation frequency in the various stages of the adenoma-carcinoma sequence changes in a gene-specific manner. Consequently, sensitive and precise diagnostic markers are needed that can be used to control early adenoma patterns for the sequence of carcinomas in the colon epithelium. Ausch et al. observed that in 75% (27 out of 36) of adenomas, ITGA4 was hypermethylated and it is a suitable biomarker for the early detection of colonic neoplasms. The ITGA4 promoter methylation is an early and recurrent phenomenon of precancerous and cancerous lesions. In addition, methylation appears in colon tissue in individuals with severe inflammation and has not been reported in any sample with a normal colon. It was obvious that during colorectal carcinogenesis, ITGA4 as putative early markers tended to be methylated slightly earlier and our meta-analysis also suggested that ITGA4 promoter methylation is a useful marker for the early diagnosis of neoplastic lesions in colitis-associated cancer.
However, the following points may be accounted as limitations of our meta-analysis. First, we observed notable heterogeneity in our results. This heterogeneity may be influenced by the difference in tumors types, ethnicity, experimental methods, and sample differences. Second, limiting the language of study to English might also induce publication bias. Third, due to the insufficient information about clinicopathological features in the included studies, the association of ITGA4 methylation with clinicopathological characteristics has not been done. Fourth, due to the limited number of studies, we pooled all tumor types in our analysis, therefore the conclusion we presented is based on the pooled data analysis and should be confirmed in the case of each cancer independetly. However, we have minimized biases by heterogeneity analysis.
Methylation of the ITGA4 gene is associated with a variety of malignancies. ITGA4 methylation has a good potential to be a possible broad-spectrum epigenetic screening marker for the diagnosis of cancers at early stages and could be effective in the future for CRC screening.
Conflict of interest statement
The authors declared no conflict of interest in this study.
R. Salehi contributed to the conceptualization; M. Yazdi contributed to data curation; S. Jafarpour and R. Nedaeinia contributed to the investigation; R. Salehi contributed to the methodology; S. Jafarpour and N. Vatandoost wrote the original draft of the article; G.A. Ferns revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.
Financial support by National Institute for Medical Research Development (NIMAD) through Grant No. 971202 is gratefully acknowledged.
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