Methylation statuses of NCOR2, PARK2, and ZSCAN12 signify tumor-infiltrating lymphocyte densities in gastric carcinoma

Tissue samples and clinicopathologic analysis

Fresh tumor tissue was obtained from three patients who underwent curative surgery for advanced gastric carcinoma (AGC) at Seoul National University Hospital, Seoul, Korea. Formalin-fixed, paraffin-embedded (FFPE) tumor material from a consecutive AGC case series (n=471) was extracted from surgical records of the Department of Pathology, Seoul National University Hospital, Seoul, Korea. Inclusion and exclusion criteria for retrospective patient selection have been described previously.23. Patients underwent gastrectomy and D2 lymph node dissection for AGC between January 2007 and December 2008. Demographics and baseline characteristics are summarized in Supplementary Table 2. Patient ages ranged from 23 to 86 years, with a average of 60.5 years, and the male to female ratio was 2.06:1. Clinical and histological data were extracted from electronic medical records, including tumor subsites in the stomach, lymph emboli, venous invasion, perineural invasion, and stage of tumor metastasis (American Joint Committee on Cancer , 7th edition). This study was approved by the Institutional Review Board of Seoul National University Hospital (Approval No. C-1803-099-931) which waived the requirement to obtain informed consent, and been conducted in accordance with the Declaration of Helsinki.

Genome-wide methylation analysis of sorted cells

Three fresh tumor tissues were enzymatically and mechanically dissociated into cell suspensions to which an APC-labeled anti-CD3 antibody and a PE-labeled anti-CD8 antibody (BD Biosciences, San Jose, CA, USA) were added. Fluorescently labeled cell suspensions were subjected to fluorescence-activated cell sorting and separated cells, including CD3–/CD8–, CD3+/CD8– and CD3+/CD8+ cells, were collected. Genomic DNA was extracted using a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), then modified with an EZ DNA Methylation Kit (Zymo Research, Irvine, CA, USA). Differences in DNA methylation between sorted cells were determined by MethylationEPIC BeadChip analysis (Illumina, San Diego, CA, USA). Array CpG probes that have a detection p-value ≥ 0.05 in more than 25% of samples were filtered out. Then, the filtered data was subjected to background correction and dye bias equalization by the R package methylumi & lumi. To reduce the bias of the Infinium I and Infinium II assays, the corrected signal value was normalized by the BMIQ (Beta Mixture Quantile) method.

Selection of DNA methylation markers that show a correlation between their methylation levels and the DNA concentrations of tumor-infiltrating lymphocytes

We sought to develop DNA methylation markers specific to two subsets, CD3+/CD8+ and CD3+/CD8− cells. The candidate MethylationEPIC probes were selected on the following criteria: (1) Δβ value > 0.78 in CD3+/CD8+ cells compared to CD3−/CD8− cells or in CD3+/CD8− cells compared to CD3−/ CD8−, (2) availability of primers and designable probes for the MethyLight assay, and (3) correlation between gene methylation levels and the proportion of CD3+/CD8+ cells or CD3+/CD8− cells.

Semi-quantitative evaluation of DNA methylation markers in gastric carcinomas

Thanks to the microscopic examination of all available tissue slides, areas up to 1 cm2 where tumor cells were most abundant and representing the most prevalent histology of the individual case were marked. Corresponding areas on unstained tissue slides were scraped with knife blades and samples were taken into microtubes containing tissue lysis buffer and proteinase K. The microtubes were held at 55°C for 24 h, incubated at 95°C for 30 min, and centrifuged. The supernatants were transferred to new microtubes. Bisulfite modification of supernatants was performed using an EZ DNA methylation kit (Zymo Research). For assessment of methylation levels at the three DNA methylation markers, the modified DNA samples were subjected to MethyLight analysis, which was performed as previously described.24. The oligonucleotide sequences of the primers and probes used for the three DNA methylation markers are listed in Supplementary Table 3.

Cell culture and reagents

Four human GC cell lines (NUGC-2, SNU-16, SNU-620 and SNU638), obtained from Korean Cell Line Bank (Seoul, South Korea) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic solution containing penicillin and streptomycin. The cells were incubated at 37°C in a humidified atmosphere at 5% CO2. Genomic DNA was extracted from these cell lines using a QIAamp DNA Mini Kit (QIAGEN).

Tissue microarray and immunohistochemical staining for CD3 and CD8

After microscopic examination, an area 2 mm in diameter was marked in the center of the tumor and two areas were marked on the invasive front. Tissue blocks were available for 453 cases, and marked areas of the corresponding tissue blocks were punched to construct a tissue microarray (TMA). Four-micrometer thick sections of TMA tissue blocks were stained with anti-CD3 antibody (1:200, Dako, Glostrup, Denmark) and anti-CD8 antibody (1:100, Neomarkers, Fremont, CA, USA). CD3 positive or CD8 positive cells were counted using open source software QuPath25. The output was CD3 TIL density and CD8 TIL density (number of cells per mm2 of tissue) of each nucleus.

statistical analyzes

Statistical analysis was performed with SPSS software for Windows, version 25.0 (SPSS, Chicago, IL, USA). To determine whether CD3 and CD8 TIL densities were normally distributed, a normality test was performed using the Shapiro-Wilk W test. P values ​​less than 0.001 were considered significant, rejecting the null hypothesis that the data was normally distributed. Spearman’s test was used to determine the correlation between variables. Recurrence-free survival (RFS) was measured from date of surgery to date of recurrence or date of death from any cause (whichever came first). The Kaplan-Meier log rank test was performed to compare overall survival (OS) and RFS times between groups. A Cox proportional hazards model was used to calculate the hazard ratio (RR) and we included in the multivariate analysis all covariates with a P-valueP-value= 0.05 as the threshold to select the final model variables. The final results are expressed in HR with 95% confidence intervals (CI).

Ethics approval

This study was approved by the Seoul National University Hospital Institutional Review Board, which waived the requirement to obtain informed consent due to the retrospective nature of this study.

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