Figure 2

Minimal spatial ITH with preferential conservation along the genome. (A) Pairwise similarity of methylomes as measured by Pearson correlation. Methylomes are more alike between spatially separated samples of the same tumor (TA-TB) and between normal colons of different patients (N-N) than between tumor and normal of the same patient (N-T) and between tumors of different patients (T-T). Correlations varied across the four groups (Kruskal-Wallis test, p < 0.0001). All between-group comparisons were strongly significant (Wilcoxon rank sum test, p < 0.0001) except for N-N vs TA-TB (p = 0.004) and N-T vs T-T (p = 0.02). (B) Comparison of methylation levels between tumor sides (TA-TB) and between different tumors for a subset of CpG sites in tumor R, r: Pearson correlation. (C) Pairwise distances (PWD) between DNA methylation of xenografts (X) after ~4 months (m) originating from a single cell were small. PWDs between parallel serial tissue culture cell lines originating from a single cell (C) were small at 2.5 months but significantly increased after 14 months (Wilcoxon rank sum test, p = 0.02). PWDs between opposite tumor sides in human tumors (TA-TB) were greater than the PWDs in the combined xenograft and cell line experiments (p = 0.04). (D) Methylation differences between glands on the same tumor side (<0.5 cm apart) and between glands on opposite sides (>1.8 cm apart) were similar. PWDs between glands within a tumor side were all greater than the four-month-old xenografts. Letters designate tumors. (E) PWDs between glands on opposite sides of the same tumor were generally greater than the PWDs of their bulks (red circles). Comparing the PWDs of glands on the same tumor side against the PWDs of the tumor bulks (black dots) revealed 4 of the 22 gland pairs with lower PWDs, possibly indicating a localized selective sweep (shaded triangle). (F) Mean PWDs between normal colons of different patients (black), between spatially separated tumor samples in the same patient (red), tumor samples from different patients (blue-white), and between normal and tumor samples from the same patient (green). Greater conservation between tumor sides (red) is seen for highly expressed genes in colorectal cancer (CRC RPKM > 1) and at promoters (TSS200). Conservation was generally greater between normal colons than between tumor sides. An exception was a subset of immune surveillance antigen processing genes that were more conserved between tumor sides than between normal colons (Wilcoxon rank sum test, p < 0.0001). The full PWD distributions are shown in Fig. S2. (G) For each tumor (n = 16), PWDs of select CpG sites are compared against the PWDs of all CpG sites. Drift is greater at non-genic (passenger) CpG sites but preferential conservation is seen at CpG sites associated with highly expressed genes in colorectal cancer (CRC RPKM > 1) and a subset of IS genes associated with antigen presentation.