Chemotherapy-resistant urothelial carcinoma has no uniformly curative therapy. Understanding how selective pressure from chemotherapy directs the evolution of urothelial carcinoma and shapes its clonal architecture is a central biological question with clinical implications. To address this question, we performed whole-exome sequencing and clonality analysis of 72 urothelial carcinoma samples, including 16 matched sets of primary and advanced tumors prospectively collected before and after chemotherapy. Our analysis provided several insights: (i) chemotherapy-treated urothelial carcinoma is characterized by intra-patient mutational heterogeneity, and the majority of mutations are not shared; (ii) both branching evolution and metastatic spread are very early events in the natural history of urothelial carcinoma; (iii) chemotherapy-treated urothelial carcinoma is enriched with clonal mutations involving L1 cell adhesion molecule (L1CAM) and integrin signaling pathways; and (iv) APOBEC-induced mutagenesis is clonally enriched in chemotherapy-treated urothelial carcinoma and continues to shape the evolution of urothelial carcinoma throughout its lifetime.
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We would like to thank our patients and their families for participation in this study. We would like to thank B. Sleckman for constructive review of the manuscript. We would also like to acknowledge D. Scherr and C. Barbieri for contributing samples, our research and clinical pathology fellows J. Fontugne, M. Kossai, C. Pauli, K. Hennrick and K. Park for their assistance during rapid autopsies, and S.S. Chae and D. Wilkes for technical assistance and constructive comments. We would also like to thank T.Y. MacDonald, J. Padilla and T. Fedrizzi for technical assistance. Work was partially supported by the Translational Research Program at WCMC Pathology and Laboratory Medicine. This work was supported by a Conquer Cancer Foundation and the John and Elizabeth Leonard Family Foundation Young Investigator Award (B.M.F.), NIH/NCATS KL2TR000458 (B.M.F.), Early Detection Research Network US NCI 5U01 CA111275-09 (J.M.M., M.A.R. and F.D.), Damon Runyon Cancer Research Foundation Clinical Investigator Award CI-67-13 (H.B.), and H2020 European Research Council ERC-CoG 648670 (F.D.).
The authors declare no competing financial interests.
Integrated supplementary information
Each row represents a gene harboring frequent SNVs (middle panel) or somatic copy number (bottom panel). Annotation rows include, from top to bottom, tumor ploidy, treatment information, biopsy site, patient’s gender, matched samples. Top bars report the otal number of non-silent SNVs in each sample. Bars on the left indicate the per-sample frequency of aberration in the cohort. NA, not available.
Per sample number of all SNVs (left boxplot) and only non-silent SNVs (right boxplot) in pre-chemotherapy and post-chemotherapy tumors. No statistical difference.
Supplementary Figure 3 Single nucleotide variants (SNV) validation by targeted sequencing (N250 panel).
Each dot represents a genomic position corresponding to an SNV sequenced with WES and with N250 approaches. Reported values are the ratios between the number of reads supporting the alternative base and the coverage. SNVs from the same patient are represented with the same color.
Supplementary Figure 4 Discordance in the SNVs between chemotherapy-naive and chemotherapy-treated UC tumors.
(a) Percentage of shared and unique SNVs in matched chemotherapy-naive and chemotherapy-treated UC tumors (same of Fig. 2a). (b) Detail of the specific amino acid change in matched chemotherapy-naive and chemotherapy-treated UC tumors. Each column represents a matched pair of pre- and post- chemotherapy tumors. Row reports the specific amino acid change. The figure highlights the wide divergence in the mutational landscape of pre- and post-chemotherapy samples.
Phylogenetic trees from 15 patients with two samples per patient.
The plot extends Fig. 5a considering samples from both WCM and TCGA cohorts. Copy number gains are represented in red and copy number losses are represented in blue. Each column represents one tumor sample. Clinical annotations include treatment, biopsy site, cohort, and the presence of TP53 SNV.
The plot shows allele specific copy number clusters as in Fig. 5a while considering samples from the TCGA cohort. Copy number gains are represented in red and copy number losses are represented in blue. Each column represents one tumor sample. Each row represents one of the 2503 genes used to compute TCGA clusters (Fig. S4.1 from Nature 507, 315–322, 2014)). Annotations include presence of TP53 SNV and TCGA clusters.
Top right scatter plot reports the clonality of SNVs in matched pre-chemotherapy and post-chemotherapy samples. Bottom and left boxplots show clonality of SNVs that are private to pre-chemotherapy and post-chemotherapy samples, respectively.
(left) Boxplot of the purity in FFPE and Fresh samples (p>0.05, Wilcoxon test). (right) Boxplot of the number of non-silent SNVs in FFPE and Fresh samples (p>0.05, Wilcoxon test).
Landscape of somatic SNVs identified.
Comparison of the percentage of shared and local SNVs in matched chemotherapy-naive and chemotherapy-treated UC tumors when considering only non-silent SNVs (top) or all SNVs (bottom).
Phylogenetic trees (top), shared and private clonally-adjusted mutations (bottom) from 6 patients with three or more tumor samples per patient.
Silent and non-silent SNVs across 12 tumor samples (top) collected at various time points and from various anatomical locations. Fractions of tumor cells harboring each mutation represented by shades of green. Reconstruction of evolutionary tree (bottom).
Supplementary Figure 14 Clonal enrichment of mutations in chemotherapy-treated UC considering silent and non-silent SNVs.
Clonal enrichment of mutations in chemotherapy-treated UC considering silent and non-silent SNVs.
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Faltas, B., Prandi, D., Tagawa, S. et al. Clonal evolution of chemotherapy-resistant urothelial carcinoma. Nat Genet 48, 1490–1499 (2016). https://doi.org/10.1038/ng.3692
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