Supplementary Figure S1 A B 7 Non Responders

Supplementary Figure S1 A B 7 Non Responders

Supplementary Figure S1 A B 7 Non Responders 4 Responders Figure S1: Extraction of paraffin-embedded PD-L1+ RCC tissues for RNA isolation. Brown staining indicates PD-L1 protein expression (IHC) in tumor foci. In (A), blue circles outline macroscopic tumor areas that were excised by manual scraping with a scalpel. In (B), focal areas of PD-L1+ tissue outlined with blue lines were excised by laser capture microdissection (LCM). Scale bars are equal to 500 um. Supplementary Figure S2 4 Relative Score p=0.36 p=0.81 p=0.32 p=0.67 p=0.46 3 2 1

0 R NR PD-1 R NR PD-L2 R NR LAG-3 R NR TIM-3 R NR Immune cell infiltrates Figure S2 legend: Molecules previously found to be up-regulated in PD-L1+ vs. PD-L1(-) melanomas are not differentially expressed in PD-L1+ RCCs from patients with divergent clinical outcomes after anti-PD-1 therapy. Expression of molecules previously found to be

associated with PD-L1 expression in melanoma (Taube et al., Clin Cancer Res 2015), and other candidate markers, were assessed by IHC in 13 PDL1+ RCC specimens, derived from 4 patients who responded to anti-PD-1 and 9 who did not. Specimens were scored for protein expression on the following scale: 0, absent expression; 1, focal expression, <5% of cells positive; 2, moderate expression, 5-50% of cells positive; 3, severe expression, >50% of cells positive. Horizontal bars indicate mean values. No significant differences were observed between responders (R) and non-responders (NR), using a two-sided Wilcoxon rank sum test. In data not shown, there were also no significant differences in FoxP3 expression or in CD4:CD8 ratios between the two groups. Gene target Supplementary Figure S3 GUSB AKR1C3 BACH2 BMP1 CACNB1 CCL3 CD24 E2F8 ENPP5 F2RL1 IL11RA KCNJ16 LTBP1 MAL MYLK2 NFATC1 PITX2 PLEC SLC23A1 SLC37A4

TNFRSF19 UCP3 UGT1A1 UGT1A3 UGT1A6 WHSC1 0 15 20 25 30 Cycle threshold 35 40 Figure S3 legend: Gene expression in RCC cell lines. The expression of 25 genes found to be differentially expressed in RCCs from anti-PD-1 responders vs. non-responders was assessed by qRT-PCR in 8 established RCC cell lines (786-0, A498, ACHN, Caki-1, RXF-393, TK-10, SN12C, and U0-31). The expression of each gene target in an individual cell line is shown as the average cycle threshold (Ct) value from triplicate reactions. Lower Ct values indicate higher gene expression. In cases of undetectable gene amplification, a Ct value of 40 was assigned (the maximum number of PCR cycles used). Vertical black bars indicate mean Ct values. The GUSB transcript was used as the internal reference. Results were visualized using GraphPad software (La Jolla, CA). RCC all stages (n=444)

FDR=0.78 0.6 High Low 0.2 B 0.0 RCC stage IV (n=71) 1.0 FDR=0.28 0.8 16 FDR =0.78 14 12 10 8 6 4 0.6 2 0.4 (n =7

1 Proportion surviving 0.4 ) 0.8 Supplementary Figure S4 expression 1.0 Relative No rm St al ( n= ag 72 e ) I (n St ag =2 1 St e II 4) ag (n =4 e 3)

I St II ( ag n= 11 e 6) IV A 0.2 0.0 Days after diagnosis Figure S4 legend: In silico analyses of TCGA RCC data do not demonstrate significant associations between UGT1A6 gene expression and overall survival or clinical stage. (A) Association of UGT1A6 expression with overall survival was assayed in silico in The Cancer Genome Atlas RCC database. A Cox regression model was used with continuous expression values of UGT1A6 in the whole patient dataset (N=444, upper panel), or only in patients with stage IV disease (n=71, lower panel). All p-values were adjusted by the Benjamini-Hochberg procedure (FDR, false discovery rate). KaplanMeier curves were generated using the median expression level to segregate samples into two groups, high and low UGT1A6 expressers.(B) The potential association of UGT1A6 mRNA expression levels in primary kidney specimens with clinical tumor stage was evaluated by fitting in a linear model using continuous expression levels of UGT1A6, and tumor stage (normal, or tumor stage I-IV) as a numeric value. The p-value adjusted by the Benjamini-Hochberg procedure (FDR, false discovery rate) is shown.

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