Volume 26, Issue 8, Pages 2241-2256.e4 (February 2019) Whole-Organ Genomic Characterization of Mucosal Field Effects Initiating Bladder Carcinogenesis  Tadeusz Majewski, Hui Yao, Jolanta Bondaruk, Woonbok Chung, Sangkyou Lee, June Goo Lee, Shizhen Zhang, David Cogdell, Guoliang Yang, Woonyoung Choi, Colin Dinney, H. Barton Grossman, Christopher Logothetis, Steven E. Scherer, Charles C. Guo, Li Zhang, Peng Wei, John N. Weinstein, Jean-Pierre Issa, Keith Baggerly, David J. McConkey, Bogdan Czerniak  Cell Reports  Volume 26, Issue 8, Pages 2241-2256.e4 (February 2019) DOI: 10.1016/j.celrep.2019.01.095 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 Clonal Enrichment of Mutations in Parallel to Evolution of Multifocal Bladder Cancer from Field Effect (A) Heatmap of all 386 non-silent mutations showing VAFs in individual mucosal samples. (B) Heatmap of VAFs in 42 genes showing variant alleles in at least 15% of the samples with VAFs ≥ 5%. (C) Density plot representing the clonality of non-silent VAFs in cluster α with a statistically significant increase in clonality with progression to HGIN and UCs. Inset shows the boxplot analysis of VAFs in three groups of samples corresponding to NU/LGIN, HGIN, and UCs. (D) Density plot representing the clonality of non-silent VAFs in cluster β with no statistically significant increase in clonality with progression to HGIN and UCs. (E) Proportion of shared mutations in individual mucosal samples. (F) Proportion of shared mutations in three groups of samples corresponding to NU/LGIN, HGIN, and UCs. For (C), (D), and (F), p values were calculated by a Kruskal-Wallis test. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 Mutagenesis Patterns as They Evolve from Field Effect (A) Composite bar graphs showing the distribution of all nucleotide substitutions in relation to cancer evolution from NU/LGIN through HGIN to UCs. It shows statistically significant increase in C > T mutations (p < 0.01) that parallel the evolution to HGIN and UCs. (B) Proportion on SNVs in specific nucleotide motif for each category of substitution in three sets of samples corresponding to NU/LGIN, HGIN, and UCs. (C) False discovery rate (FDR) for specific nucleotide motifs in progression of neoplasia from NU/LGIN through HGIN to UCs. (D) Weight scores of mutagenesis patterns in three groups of samples corresponding to NU/LGIN, HGIN, and UCs. (E) Weight scores of mutagenesis patterns in individual samples of bladder mucosa. (F) Statistical significance (p value) of mutational patterns in progression of neoplasia from NU/LGIN through HGIN to UCs. (G) Significance of contributions for mutagenesis signatures in individual samples after bootstrapping. Blue boxes indicate p < 0.05. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 Evolution of Copy Number Changes from Field Effect to Carcinoma (A) Hierarchical clustering analysis of mucosal samples according to their copy number alterations. Each column corresponds to one mucosal sample. Selected genes harboring frequent CNVs are listed on the left. (B) CNV difference calculated as Hamming distance in the three groups of samples corresponding to NU/LGIN, HGIN, and UCs. p value was calculated by a Kruskal-Wallis test. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 Whole-Organ Map of CNVs on Chromosome 7 Related to Geographic Distribution of Lesions (A) Chromosomal diagram and the CNV pattern in individual samples of a cystectomy specimen classified as NU/LGIN, HGIN, and UCs. Arrows indicate two plaque-like amplifications involving almost the entire bladder mucosa. (B) 3D pattern of CNV of chromosome 7 as it relates to the whole-organ histologic map of a cystectomy specimen shown at the bottom. (C) Enlargement of the two chromosomal regions containing plaque-like amplifications as they relate to the whole-organ histologic map shown at the bottom. (D) Genomic maps and their respective genes in two plaque-like amplified regions. Examples of CNV patterns of individual genes residing in the amplified regions with documented role in tumor pathogenesis showing their amplifications superimposed on a whole-organ histologic map are shown. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 5 Reconstruction of the Cancer Evolutionary Tree (A) Parsimony analysis showing evolutionary tree with 8 nodes of clonal expansion of successive clones in field effect corresponding to NU/LGIN with major branching at nodes four and five, one of which evolved to a separate distinct clone of HGIN. The evolution to a dominant HGIN clone occurred at node 8, and all five foci of UCs are closely related and evolved from the same HGIN clone. Clonal enrichment of VAFs for 22 genes at the transition to node 8 and the putative driver’s inactivating mutation of ACIN1 variant allele 1 (VA1) in sample E8 are indicated. It was also present in representative samples D1 and C6, corresponding to two branching subclones. The secondary inactivating mutation of ACIN1 variant allele 2 (VA2) developed in progression to HGIN. Clonal enrichment of VAFs for 22 genes at the transition to node 8 is also shown. (B) Mutational difference calculated as Hamming distance in three groups of samples corresponding to NU/LGIN, HGIN, and UCs. p value was calculated by a Kruskal-Wallis test. (C) Validation of ACIN1 mutational inactivation by Sanger sequencing with subcloning. Variant allele (VA1) and variant allele (VA2) of ACIN1 DNA tracing are shown as compared to the wild-type sequence. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 6 Evolution of Methylation Changes from Field Effect to Carcinoma (A) Hierarchical clustering using the top 57 hypo- and hypermethylated genes showing monotonic methylation change in samples corresponding to NU/LGIN through HGIN to UCs, HGIN and UCs, and UCs only. (B) Whole-organ methylation map of chromosome 19 showing chromosomal diagram and methylation pattern in individual samples of cystectomy specimen classified as NU/LGIN, HGIN, and UCs. (C) 3D pattern of hypo- and hypermethylated CpG island as it relates to the whole-organ map of a cystectomy specimen shown below. (D and E) Enlargement of the 19p (D) or 19q (E) segment showing plaque-like methylated change of individual CpG islands. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions

Figure 7 Interactive Analyses of Bladder Cancer Development from Field Effect in a Single Cystectomy Specimen with Validation in the TCGA Cohort (A) Three waves of monotonically altered pathways as disease evolves from NU/LGIN through HGIN to UCs. (B) Hierarchical clustering analysis of −log10 transformed p values for 42 pathways monotonically altered as neoplasia evolves from NU/LGIN through HGIN to UCs. (C) Hierarchical clustering showing the expression profiles of genes regulating innate immunity (inflammasome, CCR3 signaling in eosinophils, and granulocyte adhesion) in the TCGA cohort. (D) Distribution of molecular subtypes in clusters α and β shown in (C). (E) Gene set enrichment analysis showing downregulation of genes involved in innate immunity in luminal subsets of UCs in the TCGA cohort. ES, enrichment score; NES, normalized enrichment score. (F) Integrated multi-platform analysis of mutations, methylation, and CNV involved as neoplasia evolves from NU/LGIN through HGIN to UCs using iCluster. For (D), p value was calculated by a Fisher Exact test and (E) by the standard GSEA method. Cell Reports 2019 26, 2241-2256.e4DOI: (10.1016/j.celrep.2019.01.095) Copyright © 2019 The Author(s) Terms and Conditions