a suggestive example of spatial clustering of
three APOBEC-positive clones (Fig. 3J). To
study the frequency and spatial distribution
of APOBEC-positive clones, we used signature
fitting and a likelihood ratio test to annotate all
exomes according to their evidence of APOBEC
mutagenesis ( 22 ). Across donors, 22% of all
microbiopsies of normal urothelium showed
evidence of APOBEC mutagenesis (likelihood
ratio test,Q< 0.05; Fig. 3K). To determine
whether APOBEC-positive clones tend to clus-
ter in space, for each positive clone we calcu-
lated the fraction of positive clones surrounding
it (Euclidean distance <1 mm) both in the real
data and in random permutations of the data
(fig. S13) ( 22 ). This analysis suggests that
APOBEC clones appear to be scattered uni-
formly in the tissue (permutation test,P=
0.92), without evidence of spatial cluster-
ing of unrelated clones, which suggests thatAPOBEC mutagenesis is typically triggered
independently in individual cells across the
urothelium.
Copy number and rearrangement analyses
of normal urothelium revealed that the major-
ity of clones carry no structural variants ( 22 ).
Copy number alterations were detected in
only 28% of urothelial exomes, with the most
common changes involving whole or arm-
level gains of chromosomes 13, 14, 15, and 1680 2 OCTOBER 2020•VOL 370 ISSUE 6512 sciencemag.org SCIENCE
Fig. 4. The mutational landscape across histological features in patients
with bladder cancer.(AandB) Histology images depicting features microdissected
from cystectomy material for two patients with bladder cancer (C04_72M and
C03_67M). (CandD) Phylogenetic reconstruction of the evolution of cancer and
CIS clones ( 22 ). Only microbiopsies with a high degree of clonality (mean VAF≥
0.25) were included. Biopsy maps show the relative positions of macroscopic
biopsies (b01 to b10) within the bladder. Branches without a feature indicated are
histologically normal urothelium. Branch lengths depict single nucleotide variant
(SNV) counts, and the number next to each branch denotes assigned dinucleotide
variants (DNVs). Driver genes identified in this study and in previous studies
( 18 , 19 ) are annotated. Truncating mutations inEPHB1andKDM3Aare annotated
in the branch shared by the CIS and tumor for C03_67M. VBN, von Brunn’s
nest. (EtoG) Histograms showing the estimated VAF of the major clone in targeted
and exome sequencing data for three different histological features: urothelium,
von Brunn’s nests, and lymphoid aggregates. (H) Proportion of mutations located
within the B cell receptor (BCR) across histological features.RESEARCH | RESEARCH ARTICLES