Assessment of DOK7 and SPDEF as Potential Epigenetic Biomarkers in Whole Blood of Patients with Gastric Adenocarcinoma and Intestinal Metaplasia

Gastric and generally a precancerous lesion in the gastric carcinogenesis cascade. DNA methylation is one of the most deeply studied epigenetic modications. Numerous studies have been shown that aberrant methylation of DOK7, an adaptor protein, and SPDEF, a transcription factor, correlates with carcinogenesis. This study aimed to investigate DNA methylation of DOK7 and SPDEF in the whole blood specimens obtained from patients with IM and GC and normal individual controls to examine the possible implication of epigenetic biomarker for differential diagnosis GC from IM. and have signicant in and versus the Signicant methylation changes were observed for Dok7 between (p = 0.03), GC (p < cases compared to For signicant hypermethylation results obtained, for GC (p and (p = 0.03) cases in comparison to normal controls. Sensitivity and specicity for DOK7 as DNA epigenetic biomarker diagnostic of gastric cancer test determined by statistical analysis revealed high intermediate sensitivity (73.33 and high specicity (97.14 %) methylation changes with p < 0.001. For SPDEF DNA methylation test as GC biomarker by statistical analysis revealed lower sensitivity (36.67 but higher specicity (97.14 with < 0.001. The percentage of DOK7 and SPDEF promoters hypermethylation status after analysis of Ct obtained from real-time PCR test. The results of normality tests have demonstrated that methylation status has a normal distribution. IM: Intestinal Metaplasia, GC: Gastric Cancer. These results suggest that the change in the methylation of DOK7 (AUC = 0.9495) and SPDEF (AUC = 0.8419) is a promising epi-biomarker. For the rst time, this study shows blood-based biomarkers DOK7 and SPDEF genes are powerful epi-biomarkers and provide insights into gastric cancer pathogenesis and diagnosis.

These results suggest that the change in the methylation of DOK7 (AUC = 0.9495) and SPDEF (AUC = 0.8419) is a promising epi-biomarker. For the rst time, this study shows blood-based biomarkers DOK7 and SPDEF genes are powerful epi-biomarkers and provide insights into gastric cancer pathogenesis and diagnosis.

Background
Gastric Cancer, an age-related disease, is one of the most common and fatal cancer worldwide. GC incidence is highly dependent on region and culture and is observed more in men. It accounts for 783,000 deaths each year, as the third most deadly cancer worldwide [1]. The majority of GCs are associated with infectious agents, including the bacterium Helicobacter pylori and Epstein-Barr virus (EBV). Injury to the stomach's epithelial layer and in ammation, raised by Helicobacter pylori, can increase gastric stem cells' proliferation [2]. In ammation also initiates the reprogramming and proliferation of the chief cells into the intestinal cell lineage by epigenetics alterations. Metaplasia refers to representing a normal cell lineage in a tissue where it is not typically found.
Numerous challenges in the gastric cancer study's eld remained because of intratumoral, intrapatient, and interpatient heterogeneity in gastric cancer. The rst challenge is to overcome gastric cancer incidence high rate through ongoing attention to improved public health and develop precise and less invasive tools and methods for early detection. Eventually, the major challenge is translating recent discoveries in molecular biology to effective treatment for patients with gastric cancer. Therefore, precancerous lesion follow-up is advisable for secondary gastric cancer prevention [3,4].
Therefore, precancerous lesion follow-up is advisable for secondary gastric cancer prevention. Intestinaltype gastric cancer development is preceded by the development of metaplastic cell lineages in the gastric mucosa [5]. The IM prevalence rate is dramatically higher in patients with H. pylori infection. Patients with IM are at a higher risk of malignancy emergence, especially for incomplete IM and IM in the corpus [6]. In most cases, metaplastic lineages are characterized by mucus secretion [7].
Gastric carcinogenesis involves the gradual accumulation of various genetic and epigenetic alterations, leading to gain-of-function in oncogenes and loss-of-function in tumor suppressor genes. Genetic alterations, such as p53 mutations, CD44 ampli cations, and epigenetic alterations, including changes in histone modi cations, level of DNA methylation, and non-coding RNA expression, have been occurring in tumorigenesis. DNA methylation is a major mechanism of tumor-related gene inactivation, particularly tumor suppressor genes, in the neoplastic cells [8]. Accurate technologies such as whole-genome microarray analysis, immunohistochemical analysis, and DNA methylation analysis allow us to disclose intestinal metaplasia events leading to carcinogenesis.
The epigenetic biomarker's ability to detect cancer in body uids, such as serum or gastric washes, represents its noninvasive application advantages. For example, a high prevalence of alterations in the gene methylation, such as TFPI2, RUNX3, p16, and CDH1, is found in gastric cancer patients' serum [9].
Unlike tissue DNA, blood leukocyte DNA can be obtained non-invasively and inexpensively. Thus, methylation changes in blood leukocyte DNA may serve as a potential biomarker for disease diagnosis [10].
Dok7 (Downstream of Kinase 7), a peripheral membrane protein, is a Dok family member. It is a cytoplasmic activator of the muscle-speci c kinase (MuSK), involved in neuromuscular junction formation and maintenance [11]. CpG site hypermethylation was observed within the DOK7 promoter region in cancers with epithelial origins, such as breast [12] and lung [13] cancers. SPDEF (SAM Pointed Domain containing ETS transcription factor) is an ETS family transcription factor reported to playing a role in tumor progression and several cancers' metastasis. In the adult intestines, SPDEF is a direct downstream target gene of ATOH1, a master transcription factor that mediates Notchregulated differentiation of the intestinal epithelium [14]. SPDEF targets FoxM1 (Forkhead Box M1) promoter activity, which has been proven to be highly expressed in gastric cancer. Wu and colleagues found that SPDEF was overexpressed at the mRNA and protein level in human gastric cancer species [15].
In this study, two potential epigenetic biomarkers (DOK7 and SPDEF) were assessed in the blood leukocytes of patients with IM and GC and normal controls.

Data Acquisition
The relationship between DMCs of gastric tumors and non-tumoral tissue have been checked by downloading microarray data, including non-tumoral and gastric cancerous tissues, from the "NCBI GEO database" (Home -GEO -NCBI) and previous studies.

Differentially Methylated CpG (DMC) region of chosen genes and designing primers
We investigated the regulator regions from the "GeneCards database" (GeneCards -Human Genes | Gene Database | Gene Search) to identify the promoter regions. The "UCSC Genome Browser" (UCSC Genome Browser Home) was used to gain the promoters' sequences. The promoters' methylation assessment was utilized by "CpGPlot/CpGreport" (EMBOSS Cpgplot < Sequence Statistics < EMBL-EBI). We considered CpG islands with a high-frequency of CpG dinucleotides, the Obs/Exp value is greater than 0.6, and the GC content is higher than 50% characteristics. (Fig. 1) Afterward, the restriction endonuclease recognition sites are determined by the "NEBcutter analysis tool" (NEBcutter V2.0), and the speci c methylation-sensitive restriction enzymes have been chosen. Then, primer sequences are designed by "Primer3Plus" (https://primer3plus.com/) on either side of the selected restriction enzyme recognition sites. Designed Primers for amplifying the selected genes with methyl sensitive restriction enzyme polymerase chain reaction (MSRE-PCR) method are shown in Table 2.

Analysis of promoter methylation with MSRE-PCR
Promoter's CpG islands methylation changes carried out using the MSRE-PCR method. According to the manufacturer's instruction, extracted DNA was digested using the appropriate methylation-sensitive restriction enzyme (RE), SmaI for DOK7, and HhaI for SPDEF (Takara, Japan) ( Table 2). 50 ng of DNA treated with 0.5 µL of RE, 1 µL 10× universal buffer, sterile distilled water to reach the solutions' total volume to 10 µL. For undigested samples (as controls), 0.5 µL sterile distilled water was added instead of restriction enzyme. All sample tubes were incubated overnight at 37°C.
Each PCR reaction consisted of 1 µL of the DNA solution, 0.5 µL of each forward and reverse primer (with the concentration of 10 pmol/µL), and 10 µL of Taq 2× master mix, sterile distilled water to achieve a 20 µL reaction mixture for each sample. Treated DNA ampli ed by PCR using DOK7-and SPDEF-speci c primers (Table 2) and the Taq PCR Master Mix Kit (Ampliqon, Denmark).
PCR condition was performed in 30 cycles, including strings denaturation (95°C for 40"), primer annealing (60°C for 40"), extension (72°C). Also, initial denaturation set at 95°C for 5', and a nal extension set at 72°C for 7' were considered. PCR products run on the 1.5% agarose gel, stained with ethidium bromide and bands visualized under UV radiation.
To identify the accurate samples methylation, each treated sample with RE was compared with its undigested one as 100 % methylated due to no RE were used in these tubes. The intensity of the treated samples ampli ed products has a direct relationship with the methylation level. The methylation intensity was calculated by gel analyzer software (GelAnalyzer 19.1).

Quantifying with MSRE-qPCR
Real-time PCR was applied to quantify methylation alteration in DOK7 and SPDEF genes. For each sample, digested and undigested DNA samples were ampli ed. All PCRs were performed in a Rotor-Gene 6000 thermal cycler (Corbett Life Science, Australia). Real-time PCR performed with the following constituents: 1 µL of DNA solution was added to 9 µL of a PCR mixture made up of 5 µL of 2× SYBR Green PCR Master Mix (Takara, Japan), 0.2 µL of forward and reverse primers, and 4.6 µL of water. PCR condition was performed in 45 cycles, including strings denaturation (95°C for 30"), primer annealing (60°C for 30"), extension (72°C for 30"). Also, initial denaturation is set at 95°C for 5'.
ΔCt values determined as the difference between the obtained Ct values of the non-treated DNA and treated DNA (∆∆C T =∆C T(UN) -∆C T(D) ).

Statistical analysis
Results expressed as means ± standard deviation (SD), and all statistical analyses were performed using IBM SPSS version 26 (SPSS, Inc., Chicago, IL, USA) and Prism (GraphPad Software, San Diego, CA) version 9.0.0. The statistical signi cance of the difference between groups was determined using oneway ANOVA to determine the three groups' differences. P < 0.05 were considered as statistically signi cant.

Clinicopathological parameters
The present study analyzed the association between the promoter methylation status of DOK7 and SPDEF genes and clinicopathological characteristics. The patient's age range was between 27-78 years old, 41-81 years old, and 37-82 years old for normal, metaplasia, and gastric cancer cohorts, respectively. Also, 47.4 % and 52.6 % of patients were female and male. (Table 1) Differences in methylation between normal, metaplasia, and gastric cancer specimens by MSRE-PCR method In this study, the relationship between methylation patterns of DOK7 and SPDEF promoters of 60 patients with metaplasia or gastric cancer and 35 normal individuals were analyzed. Our assessments indicate that the DOK7 promoter was hypomethylated in normal samples (mean = 57.77 %) compared with IM (mean = 74 %) and GC (mean = 88.07 %) patients. Also, the methylation of the SPDEF promoter was lower in normal individuals (mean = 54.43 %) than GC (mean = 81.47%) and IM (mean = 63.13 %) ( Table 3). The difference between the methylation percentage of the DOK7 promoter in normal versus metaplasia and gastric cancer samples were signi cant (p-value < 0.001). Also, the difference in SPDEF promoter methylation in normal samples compared to metaplasia and gastric cancer patients was signi cant (pvalue < 0.001). (Fig. 2, Fig. 3) The comparison of methylated DOK7 levels in GC patients and normal controls (p-value < 0.001) revealed high intermediate sensitivity, speci city 73.33, 97.14%, respectively, with a cut-off at > 84.50 % and area under the ROC curve of 0.9495. Also, the examination of methylated SPDEF (p-value < 0.001) revealed low sensitivity and high speci city values of 36.67 and 97.14%, respectively, with a cut-off at > 89.5 and an area under the ROC curve of 0.8419 (Fig. 4).

Discussion
In this study, alterations of DOK7 and SPDEF promoter methylations were examined in DNA extracted from whole blood samples of 30 GC and 30 IM cases compared to 35 normal controls. Signi cant methylation changes were observed between cases (GC and IM) and controls. For Dok7 the level of signi cances were GC (p < 0.001), IM (p = 0.03) and for SPDEF they were GC (p < 0.001) and IM (p = 0.03) respectively. Interestingly, signi cant differences also were observed between the methylation status of GCs and IM cases themselves for DOK7 (p < 0.001) and for SPDEF (p = 0.03).
Our results conveyed that the mean of methylation in the DOK7 promoter was equal to 66 % in DNA extracted from WBCs in IM cases, whereas it was 88.07 %. In GC cases, and demonstrated signi cantly 22 % less methylation compared to IM cases. The signi cant results also were detected for SPDEF, in which more than 10 % methylation status was observed in GCs (mean 81.47 %) compared to IM (mean 71.33 %) cases.
In IM patients, the methylation status difference with normal controls was 9.17 % for DOK7 and 12.56 % for SPDEF. These results suggest that the methylation status is signi cantly elevated towards hypermethylation of DOK7 and SPDEF from normal controls to IMs and GCs, respectively.
The same results have been reported previously for DOK7 promoter methylation changes in tumor samples from breast cancer [16], esophageal squamous cell carcinoma [17], and glioma [18], in which hypermethylation of DOK7 was reported in those cancers. Moreover, a signi cant reduction in DOK7 expression has been shown in lung cancer patients, also associated with poor survival [13]. Signi cant hypermethylation of SPDEF has been reported in lung metaplasia, chronic obstructive pulmonary disease (COPD) [19].
DNA methylation changes, including local hypermethylation and global (genome-wide) hypomethylation, are associated with cancer development and tumor growth [20]. DNA hypermethylation, rationally, is not a random process and can accurately characterize the cancer status. If selected DNA methylation markers are consistent and replicable, they can be utilized in clinical practices. Since blood is a convenient tissue in noninvasive cancer assays, evaluation of cancer biomarker is under intensive attention in DNA extracted from WBC [21].
The exosomes are cell to cell communication particles secreted from a different cell type consisting of immune and cancerous cells. They contain malignant information such as nucleic acids, mRNAs or miRNAs, or proteins, such as growth factors, cytokines, chemokines, or angiogenic and immunoregulatory molecules in their cargo. These cancer-released exosomes can induce tumor growth, neovascularization, immune suppression, invasion, dissemination, and pre-metastatic niche formation. They have an important role in cancer development and metastasis [22]. Boonsongserm and colleagues demonstrated that colorectal cancer (CRC) cell exosomes induce genome-wide DNA methylation changes in the WBCs of patients with CRC [23]. Besides, several studies reported global changes in repetitive elements [24], as well as speci c CpG methylation alterations found in DNA from blood cells of patients with various cancer types [25,26].
ROC analysis was employed to compare the predictive accuracy of the methylation status of patients with gastric cancer. Our ndings demonstrated the methylation status of DOK7 (with a cut-off at > 84.50 %) could re ect the gastric lesions' malignancy.
In conclusion, this study is the rst report that revealed a signi cant alteration in DOK7 and SPDEF promoter's methylation observed in the WBCs of patients with either IM or GC as a promising tumor marker for cancer screening studies.