Figure S1-S6 from Urinary Tumor DNA MRD Analysis to Identify Responders to Neoadjuvant Immunotherapy in Muscle-invasive Bladder Cancer
Authors: Zhang Ruiyun , Zang Jingyu , Jin Di , Xie Feng , Shahatiaili Akezhouli , Wu Guangyu , Zhang Lu , Wang Lu , Zhang Yue , Zhao Zhixin , Du Pan , Jia Shidong , Fan Jinhai , Zhuang Guanglei , Chen Haige 
Published: October 2023

<p>Figure S1. (A) Representative MRI images for radiographic evaluation of neoadjuvant toripalimab. (B) Representative hematoxylin and eosin staining for histopathological evaluation of neoadjuvant toripalimab. CR = complete response; PR = partial response; SD = stable disease; PD = progressive disease. A modifier “p” refers to pathologic staging after cystectomy.

Figure S2. (A) Stacked bar plot shows the percentage of patients with negative or positive PD-L1, low or high TMB, and negative or positive TLS. (B) Oncoprint chart for the mutational landscape of tDNA in patients with ypCR or non-ypCR. Samples were analyzed by whole exome sequencing, and the mutation frequencies of each gene are shown on the right. (C) The line plot illustrates tumor size changes measured by MRI imaging before and after neoadjuvant toripalimab. PD-L1 = programmed death ligand 1; TMB = tumor mutation burden; TLS = tertiary lymphoid structure; ypCR = pathological complete response; pre-tx = pre-treatment; post-tx = post-treatment; MRI = magnetic resonance imaging.

Figure S3. (A) Oncoprint chart for the mutational landscape of tDNA and utDNA. Samples were analyzed by whole exome sequencing, and the mutation frequencies of each gene are shown on the right. (B) TMB correlation between tDNA and utDNA as assessed by whole exome sequencing. Red shading indicates 95% confidence interval. Spearman correlation coefficient (r) and P value are shown. tDNA = tumor DNA; utDNA = urinary tumor DNA; TMB = tumor mutation burden.

Figure S4. (A) Box plot compares TFsm in utDNA versus ctDNA samples collected at baseline. (B) Venn plots show the number of shared and unique variants in matched utDNA and ctDNA samples. (C) Box plot compares TFcn in utDNA versus ctDNA samples at baseline. (D) Copy number gain (red) and loss (blue) of utDNA and ctDNA, as identified by GISTIC2.0. TFsm = tumor fraction estimate based on somatic mutations; utDNA = urinary tumor DNA; ctDNA = circulating tumor DNA; TFcn = tumor fraction estimate based on copy numbers.

Figure S5. (A) The line plot illustrates TFsm changes in utDNA samples upon neoadjuvant toripalimab. (B) The line plot illustrates TFcn changes in utDNA samples before and after neoadjuvant toripalimab. (C) Stacked bar plot showed the percentage of patients with low or high pre-treatment TFsm, TFcn, and MRI measurements according to the optimal cutoff points defined by ROC analysis. (D) Stacked bar plot showed the percentage of patients with low or high post-treatment TFsm, TFcn, and MRI measurements according to the optimal cutoff points defined by ROC analysis. (E) Waterfall plot for the best change of target lesions in 20 patients. The post-treatment urinary MRD status of each patient are arranged along the x-axis. Bar color indicates the pathologic outcome of neoadjuvant toripalimab. TFsm = tumor fraction estimate based on somatic mutations; TFcn = tumor fraction estimate based on copy numbers; MRI = magnetic resonance imaging; utDNA = urinary tumor DNA; utDNA-pre = pre-treatment utDNA; utDNA-post =post-treatment utDNA; ypCR = pathological complete response; pre-tx = pre-treatment; post-tx = post-treatment; MRD = minimal residual disease.

Figure S6. (A) Spider plot indicating dynamic changes of TFsm, TFcn, and MRI measurements for each patient during neoadjuvant toripalimab. (B) Box plot illustrates utDNA reduction (defined by TFsm + TFcn < 10%) in utDNA-pre versus utDNA-post samples. (C) IGV plot showing the FGFR3 S249C mutation in patient RZ12 detected by MRD panel sequencing or whole exome sequencing. (D) VAF changes of the FGFR3 S249C mutation in patient RZ12 with progressive disease. TFsm = tumor fraction estimate based on somatic mutations; TFcn = tumor fraction estimate based on copy numbers; MRI = magnetic resonance imaging; C1 = cycle 1; C2 = cycle 2; C3 = cycle 3; C4 = cycle 4; RC = radical cystectomy; ypCR = pathological complete response; MRD = minimal residual disease; WES = whole exome sequencing; tDNA = tumor DNA; utDNA = urinary tumor DNA; utDNA-pre = pretreatment utDNA; utDNA-post = post-treatment utDNA; C2D1 = cycle 2 day 1; VAF = variant allele frequency; PD = progressive disease.</p>