Supplemental Material - Factors Associated With Postoperative Decisional Regret in Patients Undergoing Gastrointestinal Cancer Surgery: A Prospective Cohort Study

Supplemental Material for Factors Associated With Postoperative Decisional Regret in Patients Undergoing Gastrointestinal Cancer Surgery: A Prospective Cohort Study by Hiromitsu Kinoshita, Tatsuto Nishigori, Takayo Nakabe, Norihiro Shimoike, Keiko Sato, Yuichi Imanaka, Kazutaka Obama, and Yumi Matsumura in The American Surgeonâ„¢</p

Cell type-specific hypomethylation of genes involved in lineage commitment.

(A) Number of CpG sites hypermethylated or hypomethylated in one cell type of leukocytes. One CpG site was specifically hypermethylated in B cells, and three, seven, 182, and 33 CpG sites were specifically hypomethylated in CD4+ T cells, CD8+ T cells, B cells, and NK cells, respectively. (B) Lineage-specific hypomethylation of CpG sites within genes related to specific types of leukocytes. CpG sites within genes involved in B cell function, Blnk, Card11, and Cd19, were specifically hypomethylated in B cells. CpG sites within genes involved in T cell function, Cd4a, Cd8a, and Cd8b, were specifically hypomethylated in T cells. CpG sites within genes involved in NK cell function, Klrk1, Ncr1, and Pik3r1, were specifically hypomethylated in NK cells. (C) Human relevance of lineage-specific hypomethylation. The leukocyte cell type-specific hypomethylation of Cd19, Usp7, and Zbtb7b in the mouse genome was conserved at their homologous regions in the human genome.</p

The presence of DNA methylation profiles specific to individual types of mouse leukocytes.

(A) Unsupervised hierarchical clustering analysis using the top 1% (2,588 probes) of the total 258,525 probes that had the HSD. The three myeloid lineage cell types and four lymphoid lineage cell types were clustered separately. (B) Origins of the 2,588 probes against gene structure. 291 (11.3%) and 283 (10.9%) of probes were derived from enhancers and TSS200, respectively. (C) Origins of the 291 enhancer and 283 TSS200 probes against CGI. The vast majority of the HSD probes were in open sea, and 0 enhancer probe and 1 TSS200 probe were in CGI. (D) Unsupervised hierarchical clustering analysis using the top 1% (155 probes) from enhancers. The three myeloid lineages and the four lymphoid lineages were classified, similar to the use of the HSD probes from all genomic regions. (E) Unsupervised hierarchical clustering analysis using the top 1% (95 probes) from TSS200CGIs. Separation of the two lineages was not clear. (F) and (G) Principal component analysis (PCA) using the HSD probes from enhancers (F) and those from TSS200 (G). Separation of the two lineages was clearer using probes from enhancers.</p

Estimation of a leukocyte fraction in the stomach infected by <i>H</i>. <i>pylori</i> for three weeks.

(A) and 20 weeks (B) and in the DSS-treated colon (C). The fraction of infiltrating leukocytes was 1.4–9.8% in the H. pylori-infected stomach, but 0–1.4% in corresponding control samples. (PDF)</p

Comparison of two cell types using methylation levels of CpG sites within open sea and CGIs.

Using CpG sites within open sea, methylation profiles were similar between cell types from one cell lineage (A and B), but different between cell types from different cell lineages (C and D). In contrast, using CpG sites within the CGIs, methylation profiles were similar even between cell types from different cell lineages. (PDF)</p

50 CpG sites used for the estimation of the fraction of infiltrating leukocytes in the colon.

50 CpG sites used for the estimation of the fraction of infiltrating leukocytes in the colon.</p

Fraction of individual cell types among whole leukocytes.

(A) The fraction among myeloid-lineage cells. Dendric cells, monocytes, and neutrophils were separated by MACS. (B) The fraction among lymphoid-lineage cells. CD4+/CD8+ T cell, B cells, and NK cells were separated by MACS. (PDF)</p

Selection of CpG sites methylated in all types of leukocytes and unmethylated in gastrointestinal tissues (probes).

(A) From the total 258,525 CpG sites, 14,186 CpG sites highly methylated (β ≥ 0.9) in the total leukocyte samples (n = 2) were selected. From these 14,186 CpG sites, the top 50 CpG sites with lowest methylation levels in the gastric epithelium (average methylation levels of 4 mice) or colonic epithelium (average methylation levels of 2 mice) were selected as leukocyte fraction markers in the stomach and colon, respectively. (B) DNA methylation levels of isolated marker CpG sites in individual types of leukocytes. Marker probes were also highly methylated in all the individual cell types of leukocytes. (C) To estimate a fraction of leukocytes in a DNA sample, DNA methylation levels of 50 CpG markers were plotted [x = Δβ value (leukocytes–non-inflamed epithelium); y = Δβ value (inflamed epithelium–non-inflamed epithelium)]. The 50 CpG markers can be classified into 1) markers whose methylation levels were not affected by chronic inflammation (left) and 2) markers whose methylation levels were affected by chronic inflammation (middle). Therefore, a regression line whose slope was considered as the leukocyte fraction was drawn using markers not affected by chronic inflammation (right). 40 of 50 CpG markers were utilized as those not affected by chronic inflammation. (PDF)</p

Comparison of methylation levels of all genomic regions, TSS200CGIs, and enhancers between individual types of leukocytes.

Using all the genomic regions and the enhancers, methylation profiles were similar between cell types in one cell lineage (A and B), but different between cell types from different cell lineages (C and D). In contrast, methylation profiles were similar even between cell types from different cell lineages in the TSS200CGIs. The x and y axes show DNA methylation levels of individual cell types.</p

Diagrammatic summary of the establishment and validation of an estimation algorithm using methylation marker CpG sites to estimate the leukocyte fraction.

Diagrammatic summary of the establishment and validation of an estimation algorithm using methylation marker CpG sites to estimate the leukocyte fraction.</p