Additional figures and tables from TFAP2E Methylation and Expression Status Does Not Predict Response to 5-FU-based Chemotherapy in Colorectal Cancer

Supplementary data Supplementary Methods Supplementary Table 1. Characteristics of Epicolon I patients (n=532) Supplementary Table 2. Characteristics of Clinical cohort patients (n=251) Supplementary Table 3. Primer sequences Supplementary Figure 1. Schematic representation of TFAP2E gene. The gene encoding transcription factor AP-2 epsilon (TFAP2E) is located on chromosome 1p34. In the schematic overview, exons are represented by blue boxes and introns with blue lines. The TFAP2E gene has two cytosine-phosphate-guanine (CpG) islands underscoring the potential of gene expression by means of CpG hypermethylation (green boxes). We used two quantitative methods, QMSP and pyrosequencing, covering both CpG islands in order to correlate methylation levels at these two locations. Supplementary Figure 2. Methylation analysis of TFAP2E CpG islands in CRC tissues. Calculation of cutoff value of TFAP2E Intron 3 methylation. Results comparing methylation of the TFAP2E CpG1 (Promoter/Exon 1) and CpG2 (Intron 3) in CRC tissues (N=50) by (A) Bisulfite pyrosequencing (% Methylation) (B-C) Bisulfite pyrosequencing results of TFAP2E Intron 3 methylation in normal colon mucosa from healthy subjects N-N (N=21); and CRC patients-matched tumor-and non-tumor samples N-C (N=41);(N=41). (D) Whitin-subject Receiver Operating Characteristics (ROC) analysis performed on measured TFAP2E % (percentage of methylation) values of patient-matched tumor- (N-N; N=21) and non-tumor (N-C; N=41) samples, respectively. The area under the ROC-curve was calculated as 0.73 (95% CI:0.62-0.82), indicating that the probability of observing a higher TFAP2E value in a tumor-sample of a patient compared to its related non-tumor sample is about 73%. Supplementary Figure 3. Correlation between TFAP2E intron 3 methylation and IHC expression. When methylation was analysed as a continuous variable using linear regression analysis, we observed a trend towards negative correlation with IHC protein expression; however, this correlation was significant only when combining lack of and low TFAP2E expression (IHC scores 0 & 1) versus strong TFAP2E staining (IHC scores 2 & 3) (p= 0.015). Linear regression analysis comparing TFAP2E methylation and expression in CRC tissues (N=314) using IHC results by binary scoring system: positive/negative (A) staining scores 0 & 1 vs 2 & 3(B) and the staining intensity scale 0 to 3 (C). Correlation and performance measures between TFAP2E methylation and expression in CRC tissues using IHC and methylation qualitative results (staining scores 0 & 1 vs 2 & 3; (D). Supplementary Figure 1. Schematic representation of TFAP2E gene. The gene encoding transcription factor AP-2 epsilon (TFAP2E) is located on chromosome 1p34. In the schematic overview, exons are represented by blue boxes and introns with blue lines. The TFAP2E gene has two cytosine-phosphate-guanine (CpG) islands underscoring the potential of gene expression by means of CpG hypermethylation (green boxes). We used two quantitative methods, QMSP and pyrosequencing, covering both CpG islands in order to correlate methylation levels at these two locations. Supplementary Figure 2. Methylation analysis of TFAP2E CpG islands in CRC tissues. Calculation of cutoff value of TFAP2E Intron 3 methylation. Results comparing methylation of the TFAP2E CpG1 (Promoter/Exon 1) and CpG2 (Intron 3) in CRC tissues (N=50) by (A) Bisulfite pyrosequencing (% Methylation) (B-C) Bisulfite pyrosequencing results of TFAP2E Intron 3 methylation in normal colon mucosa from healthy subjects N-N (N=21); and CRC patients-matched tumor-and non-tumor samples N-C (N=41);(N=41). (D) Whitin-subject Receiver Operating Characteristics (ROC) analysis performed on measured TFAP2E % (percentage of methylation) values of patient-matched tumor- (N-N; N=21) and non-tumor (N-C; N=41) samples, respectively. The area under the ROC-curve was calculated as 0.73 (95% CI:0.62-0.82), indicating that the probability of observing a higher TFAP2E value in a tumor-sample of a patient compared to its related non-tumor sample is about 73%. Supplementary Figure 3. Correlation between TFAP2E intron 3 methylation and IHC expression. When methylation was analysed as a continuous variable using linear regression analysis, we observed a trend towards negative correlation with IHC protein expression; however, this correlation was significant only when combining lack of and low TFAP2E expression (IHC scores 0 & 1) versus strong TFAP2E staining (IHC scores 2 & 3) (p= 0.015). Linear regression analysis comparing TFAP2E methylation and expression in CRC tissues (N=314) using IHC results by binary scoring system: positive/negative (A) staining scores 0 & 1 vs 2 & 3(B) and the staining intensity scale 0 to 3 (C). Correlation and performance measures between TFAP2E methylation and expression in CRC tissues using IHC and methylation qualitative results (staining scores 0 & 1 vs 2 & 3; (D). Supplementary Figure 4. Epicolon I. Overall Survival Stage IV Patients: TFAP2E IHC Expression. Disease Free Survival Stage II and III Patients: TFAP2E IHC Expression. (A) Overall survival of patients with stage IV disease, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression;). (B) Overall survival of patients that received or did not receive (C) 5-FU based chemotherapy according to TFAP2E expression status. (D) Disease Free Survival (DFS) of patients with stage II and III CRC, according to TFAP2E expression status comparing staining scores 0 & 1 (Low expression) vs staining scores 2 & 3 (High expression) (E) DFS of stages II+III CRC patients that received or did not receive (F)) adjuvant chemotherapy according to TFAP2E expression status.