High systemic and tumor-associated IL-8 correlates with reduced clinical benefit of PD-L1 blockade

Abstract

Although elevated plasma interleukin-8 (pIL-8) has been associated with poor outcome to immune checkpoint blockade ¹, this has not been comprehensively evaluated in large randomized studies. Here we analyzed circulating pIL-8 and IL8 gene expression in peripheral blood mononuclear cells and tumors of patients treated with atezolizumab (anti-PD-L1 monoclonal antibody) from multiple randomized trials representing 1,445 patients with metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma. High levels of IL-8 in plasma, peripheral blood mononuclear cells and tumors were associated with decreased efficacy of atezolizumab in patients with mUC and metastatic renal cell carcinoma, even in tumors that were classically CD8⁺ T cell inflamed. Low baseline pIL-8 in patients with mUC was associated with increased response to atezolizumab and chemotherapy. Patients with mUC who experienced on-treatment decreases in pIL-8 exhibited improved overall survival when treated with atezolizumab but not with chemotherapy. Single-cell RNA sequencing of the immune compartment showed that IL8 is primarily expressed in circulating and intratumoral myeloid cells and that high IL8 expression is associated with downregulation of the antigen-presentation machinery. Therapies that can reverse the impacts of IL-8-mediated myeloid inflammation will be essential for improving outcomes of patients treated with immune checkpoint inhibitors.

Extended Data Fig. 1. Study profile of IMvigor210, IMvigor211 and IMmotion150 trials

a, Study profile of IMvigor210, IMvigor211 and IMmotion150 trials. Flowchart showing number of intent-to-treat (ITT) patients IMvigor210, IMvigor211 and IMmotion150, as well as the numbers of patients whose plasma, PBMC and RNAseq samples were included for analysis. Tables showing the demographic characteristics of biomarker-evaluable patients in b, IMvigor210 (n=329) and IMvigor211 (n=868) cohorts, and c, IMmotion150 (n=248) cohort. P values are calculated by two-sided Fisher’s exact test. Tables showing univariate and multivariate logistic regression analyses of baseline plasma IL8 with different factors in overall survival in d, IMvigor210 (n=329) and IMvigor211(n=868) cohorts and e, IMmotion150 (n=248) cohort. HR calculated using stratified Cox proportional hazard regression models, and P values calculated using stratified log-rank test (for details, see Methods). P values were adjusted for multiple comparisons. Multivariate analyses adjusted HRs for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD) in mUC; and age, sex, Memorial Sloan Kettering Cancer Risk (MSKCC) prognostic risk score, previous nephrectomy, and SLD in mRCC data sets.

Extended Data Fig. 2. Correlation between pIL8 and other cancer immunotherapy biomarkers

a, Correlation between pIL8 and neutrophil-to-lymphocyte ratio (NLR) in mUC (IMvigor210) (n=217). NLR (x-axes) were log10 transformed before Pearson correlations (Corr) with pIL8, which were log 2 transformed (y-axes). The corresponding p values (two tailed t-test) are shown. Correlation between pIL8 and b, T-effector (Teff) (n=329), c, Tumor mutation burden (TMB) (n=255), d, Neoantigen load (n=230), e, PD-1 expression (PDCD1), f, PD-L1 (CD274), g, TGFb-response (F-TBRS), h, Tumor immune dysfunction and exclusion (TIDE) T cell dysfunction signature (n=329). Pearson correlations (Corr) between labeled biomarkers (x-axes) with pIL8, which were log 2 transformed (y-axes) and corresponding p values (two tailed t-test) are shown. i, Microsatellite instable (MSI) status. MSS, microsatellite stable; MSI-H, microsatellite instable-high (n=329).

Extended Data Fig. 3. Elevated baseline pIL8 is associated with poor clinical outcome

a, Kaplan-Meier curves depict overall survival (OS) of median T-effector (Teff) signature in cohort 2 of IMvigor210. Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated using stratified Cox proportional hazards regression models, and p values were calculated using stratified log-rank test. HR and p value are adjusted for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD). b, Kaplan-Meier curves depict overall survival (OS) of baseline plasma IL8 (pIL8) levels in cohort 1 of IMvigor210. Censored data are indicated by vertical tick marks in Kaplan-Meier curves. Number of patients per group and time point are indicated below the graphs. Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated using stratified Cox proportional hazards regression models, and p values were calculated using stratified log-rank test. HR and p value are adjusted for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD). c, Association between high vs low pIL8 (median cutoff) and Objective Response Rate (ORR) in cohort 1 of IMvigor210. High baseline pIL8 levels were associated with a higher number of nonresponders (SD and PD) (P= 0.025, two-sided Fisher’s exact test) by Response Evaluation Criteria in Solid Tumors (RECIST) 2.1. [CR: complete response; PR, partial response; SD, stable disease; PD, progressive disease]. d, Association between high vs low Teff (median cutoff) and OS in cohort 2 of IMvigor210 (HR: 0.71, 95% CI: 0.53, 0.95, P=0.0201). Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated using stratified Cox proportional hazards regression models, and p values were calculated using stratified log-rank test. HR and p value are adjusted for sex, age, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD). e, Association between high vs low baseline plasma IL8 (median cutoff) and Objective Response Rate (ORR) in IMmotion150A trend in low plasma IL8 in Atezo monotherapy associated with a higher number of responders compared to Atezo+Bev and Sunitinib treatment arms (CR and PR) (P= 0.348, 0.409 and 0.271, respectively two-sided Fisher’s exact test) f, Association of high vs low of baseline tumor IL8 expression and Objective Response Rate (ORR) in IMmotion150. A trend observed in low tumor IL8 in atezo monotherapy associated with higher numbers of responders (CR and PR) compared to Atezo+Bev and Sunitinib treatment arms (P= 0.178, 0.05, and 0.773, respectively).

Extended Data Fig. 4. Elevated on-treatment pIL8 is associated with poor clinical outcome

Association between high vs low ratio of pIL8 levels on treatment cycle 3 day 1 (C3D1) and baseline (C1D1) and Objective Response Rate (ORR) in a, cohort 1, b, cohort 2 of IMvigor210. High ratios were associated with a higher number of nonresponders (SD and PD) in cohort 1 (P= 0.042, two-sided Fisher’s exact test) and cohort 2 (P= 0.027, two-sided Fisher’s exact test) of IMvigor210. c, Kaplan-Meier curves depict OS of C3D1 and C1D1 ratio of pIL8 levels in cohort 1 of IMvigor210 (HR: 4.98, 95% CI: 1.83, 13.5, P=0.0016). Hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated using stratified Cox proportional hazards regression models, and p values were calculated using stratified log-rank test. HR and p value are adjusted for sex, age, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD). d, High ratios were significantly associated with a higher number of nonresponders (SD and PD) in atezolizumb (P=8.22e-4, two-sided Fisher’s exact test) and but not in chemotherapy (P=0.060, two-sided Fisher’s exact test) arms of IMvigor211. e, The absolute lymphoid and myeloid counts in patients in atezolizumb (n=443) and chemotherapy (n=425) arms IMvigor211. Absolute lymphocyte counts: Atezo, C1D1: minima: 0, maxima: 3.74, Percentile 75%: 1.70, 50%: 1.32, 25%: 1.00. Atezo, C3D1: minima: 0, maxima: 3.5, Percentile 75%: 1.75, 50%: 1.38, 25%: 1.00. Chemo, C1D1: minima: 0, maxima: 4.08, Percentile 75%: 1.70, 50%: 1.20, 25%: 0.90. Chemo, C3D1: minima: 0, maxima: 3.89, Percentile 75%: 1.81, 50%: 1.40, 25%: 1.00. Absolute monocyte counts: Atezo, C1D1: minima: 0, maxima: 2.25, Percentile 75%: 0.80, 50%: 0.61, 25%: 0.49. Atezo, C3D1: minima: 0, maxima: 2.2, Percentile 75%: 0.82, 50%: 0.61, 25%: 0.49. Chemo, C1D1: minima: 0, maxima: 2.2, Percentile 75%: 0.82, 50%: 0.61, 25%: 0.49. Chemo, C3D1: minima: 0, maxima: 3.08, Percentile 75%: 0.90, 50%: 0.70, 25%: 0.50. P values are calculated by two-sided Mann-Whitney U-tests.

Extended Data Fig. 5. Single cell RNAseq profiles of PBMC from bladder patients in IMvigor210 trial

UMAP plot of the mUC PBMCs, with each cell in the entire single cell RNAseq color coded for (left to right): a, responses (Responders (n=7903) and nonresponders (n=6571)); b, the corresponding patient R1 (n=2761), R2 (n=1522), R3 (n=463), R4 (n=1194), R5 (n=1963) and NR1 (n=3189), NR2 (n=849), NR3 (n=1018), NR4 (n=697), NR5 (n=818) and c, the number of transcripts detected in that cell (log 10 scale). d, the fraction of cells originating from responders and nonresponders; e, the fraction of cells originating from each of the 10 patients; f, box plots of the number of transcripts (log10) across all different cell types. Monocytes (n=6761), minima: 2.92, maxima: 4.35, and Percentile 75%: 3.59, 50%: 3.39, 25%: 3.20. CD16 Monocytes (n=623), minima: 2.92, maxima: 3.65, and Percentile 75%: 3.29, 50%: 3.23, 25%: 3.16. DC-like (n=305), minima: 2.96, maxima: 4.17, and Percentile 75%: 3.55, 50%: 3.35, 25%: 3.19. DC (n=391), minima: 2.95, maxima: 3.65, and Percentile 75%: 3.24, 50%: 3.17, 25%: 3.09. Megakaryocyte (n=294), minima: 2.94, maxima: 3.67, and Percentile 75%: 3.67, 50%: 3.19, 25%: 3.11. CD8+ T cells (n=565), minima: 2.93, maxima: 4.15, and Percentile 75%: 3.61, 50%: 3,42, 25%: 3.21. CD8+ Tcm (n=1388), minima: 2.93, maxima: 4.22, and Percentile 75%: 3.51, 50%: 3.34, 25%: 3.19. CD8+ Tem (n=1194), minima: 2.92, maxima: 4.10, and Percentile 75%: 3.56, 50%: 3,38, 25%: 3.20. CD4+ T cells (n=443), minima: 2.94, maxima: 4.02, centre: 0.75 and Percentile 75%: 3.58, 50%: 3.39, 25%: 3.21. CD4+ Tcm (n=451), minima: 2.91, maxima: 4.45, and Percentile 75%: 3.54, 50%: 3.32, 25%: 3.18. CD4+ Tem (n=335), minima: 2.93, maxima: 4.06, and Percentile 75%: 3.47, 50%: 3.28, 25%: 3.15. Tregs (n=238), minima: 2.90, maxima: 4.15, and Percentile 75%: 3.49, 50%: 3.30, 25%: 3.14. NK cells (n=1099), minima: 2.90, maxima: 4.27, and Percentile 75%: 3.56, 50%: 3.36, 25%: 3.16. B cells (n=387), minima: 2.91, maxima: 3.96, and Percentile 75%: 3.51, 50%: 3.32, 25%: 3.19, g, UMAP plot shows the distribution of different cell types between responders and non responders. Responders (n=7903) and nonresponders (n=6571).

Extended Data Fig. 6. Differential expression analysis of IL8 expression in myeloid cells

a, Gene set enrichment REACTOME pathways analysis between IL8 high vs IL8 low cells (median cutoff) in all myeloid clusters (n=8374). Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. Normalized enrichment scores, log FDR corrected, are shown in x axis. Top 10 pathways associated with IL8 high myeloid cells were shown in orange and top 10 pathways associated with IL8 low myeloid cells were shown in blue. b, Differential gene expression of IL8 high vs low populations in different myeloid clusters: a, Monocytes (n=6761), b, CD16 Monocytes (n=623), c, DC (n=391), d, DC-like (n=305) and e, Megakaryocytes (n=294). Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. Genes that are enriched in IL8 high are shown in orange and those that are enriched in IL8 low are shown in blue.

Extended Data Fig. 7. Differential expression analysis of response in myeloid cells

a, Differential gene expression analysis between responders (n=3988) and non responders (n=4386) in all myeloid cells shows a significant enrichment of myeloid inflammatory response genes (red) in non responders whereas a significant enrichment of antigen presentation machinery genes (green) in responders. b, Gene set enrichment REACTOME pathways analysis between responders and non responders in myeloid cells. Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. Normalized enrichment scores, log FDR corrected, are shown in x axis. Top 10 pathways associated with responders were shown in green and top 10 pathways associated with non responders were shown in red. Differential gene expression analysis between responders and non responders within c, Monocytes (n=6761), d, CD16 Monocytes (n=623), e, DC (n=391), f, DC-like (n=305) and g, Megakaryocytes (n=294) shows a significant enrichment of myeloid inflammatory response genes (red) in non responders whereas a significant enrichment of antigen presentation machinery and T cell activation genes (green) in responders. Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections.

Extended Data Fig. 8. Differential expression analysis of IL8 expression in tumor associated myeloid cells from single cell RNAseq of RCC patients

UMAP plot of the mRCC blood and tumor, with each cell in the entire single cell RNAseq color coded for (left to right): a, Blood (n=13,694) and Tumor (n=11,765); b, the corresponding patient c, the proportion of cells identified in each cell type in blood and tumor d, the proportion of cells identified in each cell type in each patient. e, Scaled average expression of cell type specific markers in scRNA of mRCC tumor. Tcm, central memory T cell; Tem, effector memory; M1-like, M1-like macrophages; M2-like, M2-like Macrophages. Differential gene expression analysis between IL8-high and IL8-low within each myeloid cell type in the tumor. f, monocytes (n=2821). g, M1-like macrophages (n=2452). h, M2-like macrophages (n=553). i, CD16 monocytes (n=454) shows a significant enrichment of myeloid inflammatory response genes (orange) in IL8 high whereas a significant enrichment of antigen presentation machinery genes (blue) in IL8 low. Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections.

Extended Data Fig. 9. Correlation of IL8 gene expression and neutrophil score in bladder and RCC tumors

IL8 gene expression and histological assessment of neutrophils by H&E stain in a, mRCC (IMmotion 150) (n=100) tumors. Neutrophil score 0 (n=37): minima: −2.19, maxima: 4.07, and Percentile 75%: 1.63, 50%: 0.89, 25%: 0.04. Neutrophil score 1 (n=24): minima: −2.21, maxima: 7.62, and Percentile 75%: 3.8, 50%: 1.5, 25%: 0.10. Neutrophil score 2 (n=18): minima: −2.54, maxima: 7.72, and Percentile 75%: 6.63, 50%:4.45, 25%:2.6. Neutrophil score 3 (n=21): minima: −1.96, maxima:12.6, and Percentile 75%: 7.20, 50%: 6.46, 25%: 5.49. P values are calculated by two-sided Mann-Whitney U-tests with Benjamini & Hochberg corrections. b, mUC (IMVigor 210) (n=339) tumors. Neutrophil score 0 (n=227): minima: −2.67, maxima: 2.58, and Percentile 75%: 0.25, 50%: −0.34, 25%: −0.89. Neutrophil score 1 (n=39): minima: −1.06, maxima: 1.95, and Percentile 75%: 0.70, 50%: 0.18, 25%: −0.38. Neutrophil score 2 (n=33): minima: −0.63, maxima: 2.19, and Percentile 75%: 0.97, 50%: 0.50, 25%: 0.39. Neutrophil score 3 (n=40): minima: −0.99, maxima: 2.83, and Percentile 75%: 1.60, 50%: 1.27, 25%: 0.92. P values are calculated by two-sided Mann-Whitney U-tests with Benjamini & Hochberg corrections. Neutrophils were identified by trained pathologists based on their unique morphological features. Prevalence of neutrophils was graded on a scale from 0 to 3 as follows: 0 – absence of neutrophils, 1 – rare neutrophils, 2 – moderate number of neutrophils, 3 – numerous neutrophils in the form of large aggregates or sheets. c, Representative images of 22 out of a total of 59 samples examined (37%) showing positive signals of IL8 in situ hybridization (ISH; green signal, left panel) and H&E-stain (right panel) of sections from the same area of the specimen. ISH shows IL8 expression in tumor and myeloid cells; H&E shows neutrophils (yellow arrows) in the vicinity of IL8 expressing cells.

Extended Data Fig. 10. Differential gene expression in high and low pIL8 in CD8 T cell clusters and in PBMC from the entire IMvigor210 cohort

a, Differential single cell RNAseq gene expression of CD8 T cell clusters from plasma IL8 high (n=5) vs low patients (n=5). Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. b, Differential NanoString gene expression of plasma IL8 high vs low patients in IMvigor210 (n=407). Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections.

Figure 1.. Plasma IL8 and clinical outcomes in metastatic urothelial carcinoma (mUC) and metastatic renal cell carcinoma (mRCC)

a, High baseline plasma IL8 (pIL8) levels (median cutoff: 15 pg/mL) were significantly associated with worse overall survival (OS) in cohort 2 of IMvigor210 (HR=1.84, 95% CI: 1.27, 2.66, P=1.2e-3). b, High baseline pIL8 levels were associated with a higher number of nonresponders (SD and PD) (P= 0.013, two-sided Fisher’s exact test) by Response Evaluation Criteria in Solid Tumors (RECIST) 2.1. [CR: complete response; PR, partial response; SD, stable disease; PD, progressive disease]. c, High baseline pIL8 was associated with poor OS in tumors with a T effector infiltrate signature (CD8A, GZMA, GZMB, PRF1) in mUC patients in cohort 2 of IMvigor210 (HR: 1.84; 95% CI: 1.27, 2.66, P=0.010). d, In a randomized mUC Phase 3 trial, IMvigor211, high baseline pIL8 levels were associated with worse OS in both the atezolizumab (HR: 1.84; 95% CI: 1.8, 2.26, P=4.74e-5) and chemotherapy (HR: 1.67; 95% CI: 1.38, 2.03, P=1.08e-7) treatment arms. e, Kaplan–Meier curves depict median overall survival in the atezolizumab (atezo) + bevacizumab (bev), atezolizumab monotherapy, and sunitinib treatment arms. High baseline pIL8 was associated with worse OS in the atezolizumab (HR: 2.55, 95% CI: 1.18, 5.5, P=0.017) arm but not atezolizumab + bevacizumab (HR: 1.25, 95% CI: 0.61, 2.60, P=0.535) and sunitinib arm (HR: 1.48, 95% CI: 0.69, 3.20, P=0.314) in a randomized mRCC Phase 2 trial, IMmotion150. HRs in Figure 1a,c-e were calculated using stratified Cox proportional hazard regression models, and P values were calculated using stratified log-rank test. P values were adjusted for multiple comparisons. Multivariate analyses adjusted HRs for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD) in mUC; age, sex, Memorial Sloan Kettering Cancer Risk (MSKCC) prognostic risk score, previous nephrectomy, and SLD in mRCC data sets.

Figure 2.. On-treatment changes in plasma IL8 and overall survival (OS) in mUC patients treated with atezolizumab or chemotherapy

a, On-treatment changes in plasma IL8 (pIL8) are expressed as the ratio of pIL8 level on treatment cycle 3 day 1 (C3D1) and baseline (cycle 1 day 1, C1D1). High on-treatment increases in pIL8 (median cutoff: 1.09 pg/mL) were significantly associated with worse OS in cohort 2 of IMvigor210 (HR: 2.09, 95% CI: 1.32, 3.30, P=1.55e-3). b, High on-treatment increase in pIL8 was significantly associated with worse OS in the atezolizumab arm (HR: 2.01, 95% CI: 1.49, 2.71, P=6.09e-6), but not in the chemotherapy arm of IMvigor211 (HR: 1.17, 95% CI: 0.89; 1.55, P=0.27). The HRs were calculated using stratified Cox proportional hazard regression models, and P values were calculated using stratified log-rank test. P values were adjusted for multiple comparisons. Multivariate analyses adjusted HRs for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (SLD).

Figure 3.. Poor clinical outcome and lower expression of antigen presentation genes associated with IL8-high myeloid subsets in PBMCs

a, Different cell types showing subsets of distinct myeloid and lymphoid clusters, as revealed by single cell RNAseq of baseline peripheral blood mononuclear cells (PBMC) isolated from n=5 responders and n=5 nonresponders in mUC IMvigor210 cohort. Cell types were verified based on expression of the lymphocytic (upper panel) (n=6100) and myeloid (lower panel) (n=8374) cell type-specific markers shown in b. c, UMAP plot showing the expression of IL8 in all cell clusters in responders (n=7903) and non responders (n=5671). d, Split violin plot showing the expression of IL8 and cell numbers of different myeloid and lymphoid cell types between responders and nonresponders. (Two-sided Mann-Whitney U-tests with Benjamini & Hochberg corrections) ***FDR <0.001; **FDR<0.01. e, Differential expression of IL8 high vs low populations (by median of all myeloid cells) in combined myeloid clusters. Monocytes (n=6761), minima: 0, maxima: 7.87, and Percentile 75%: 5.37, 50%: 3.99, 25%: 2.83. CD16 Monocytes (n=623), minima: 0, maxima: 4.09, and Percentile 75%: 2.31, 50%: 1.53, 25%: 0.45. DC-like (n=305), minima: 0, maxima: 5.70, and Percentile 75%: 2.68, 50%: 1.60, 25%: 0.92. DC-like (n=391), minima: 0, maxima: 5.73, and Percentile 75%: 2.23, 50%: 1.75, 25%: 0.98. Megakaryocyte (n=294), minima: 0, maxima: 6.09, and Percentile 75%: 3.67, 50%: 2.86, 25%: 2.12. CD8+ T cells (n=565), minima: 0, maxima: 3.56, and Percentile 75%: 1.31, 50%: 2.73, 25%: 0.87. CD4+ T cells (n=443), minima: 0, maxima: 5.19, and Percentile 75%: 0.76, 50%: 0.63, 25%: 0.48. Tregs (n=238), minima: 0, maxima: 3.72, and Percentile 75%: 1.23, 50%: 1.07, 25%: 0.95. NK cells (n=1099), minima: 0, maxima: 3.80, and Percentile 75%: 1.56, 50%: 1.32, 25%: 1.16. B cells (n=387), minima: 0, maxima: 3.84, and Percentile 75%: 1.84, 50%: 1.36, 25%: 1.27. Differential gene expression analysis between IL8-high (n=4187) and -low (n=4187) myeloid cells showing enriched expression of myeloid inflammatory response genes (orange) in IL8 high myeloid cells versus higher expression of antigen presentation genes (blue) in IL8 low myeloid cells. Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. f, Kaplan-Meier curves depict overall survival of IL8 expression in PBMC of IMvigor210 using median cutoff. High IL8 gene expression in PBMCs was significantly associated with worse OS in mUC IMvigor210 (HR: 1.36, 95% CI: 1.06, 1.73, P=0.014). g, Kaplan-Meier curves depict overall survival of IL8 expression in PBMC of IMvigor211 using median cutoff in atezolizumab and chemo arms, respectively. High IL8 gene expression in PBMCs was significantly associated with worse OS (HR: 1.41, 95% CI: 1.12, 1.79, P=0.0038) in atezolizumab arm, but not chemotherapy arm (HR: 0.85, 95% CI: 0.67, 1.09, P=0.214). h, Kaplan-Meier curves depict overall survival of IL8 expression in PBMC of IMmotion using median cutoff in atezolizumab, atezolizumab + bevacizumab and sunitinib arms. High IL8 gene expression in PBMCs was associated with worse OS in the atezolizumab arm (HR 2.89; 95% CI 1.16, 7.2, P=0.023) of mRCC patients in IMmotion150 but not in atezolizumab + bevacizumab (HR 1.21; 95% CI: 0.57, 2.6, P= 0.624) or sunitinib (HR: 1.20; 95% CI: 0.58, 2.5, P= 0.620) arms. HRs in Figure 3f-h were calculated using stratified Cox proportional hazard regression models, and P values were calculated using stratified log-rank test. P values were adjusted for multiple comparisons. Multivariate analyses adjusted HRs for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD) in mUC; age, sex, Memorial Sloan Kettering Cancer Risk (MSKCC) prognostic risk score, previous nephrectomy, and SLD in mRCC data sets.

Figure 4.. Single-cell RNASeq analysis of IL8 gene expression in immune subsets from matched intratumoral and peripheral blood leukocytes from RCC patients and association of tumor IL8 gene expression with clinical outcomes in mUC and mRCC

a, Single cell RNAseq (scRNAseq) identifies immune cells subsets in leukocytes in four matched blood and tumor from mRCC. UMAP plot of 25,459 immune cells, color-coded by their annotation from four matched blood and tumor (single cell n= 13694 and 11765 cells, respectively). Color-coded expression (grey to red) of the lymphocytic (left two panels) and myeloid (right two panels) cell type-specific markers from four matched blood and tumor shown in b. c, Heat map reporting scaled expression (log expression count values) of selected gene sets. Gene expression color scheme is based on scale log count expression distribution, from –2.0 (blue) to 2.0 (red). Color bars in right margin highlight cell subsets of interest. d, Differential gene expression analysis between IL8-high and -low intratumoral myeloid cells showing enriched expression of myeloid inflammatory response genes (orange) in IL8 high myeloid cells versus higher expression of antigen presentation genes (blue) in IL8 low myeloid cells. Volcano plot representing differentially expressed (FDR-corrected p<0.05) between IL8 high and IL8 low myeloid cells in the four mRCC tumors (n= 6280 cells). Differential expression analysis with the generalized linear models (glm)-based statistical methods of the edgeR package with Benjamini & Hochberg corrections. e. High vs. low intratumoral IL8 gene expression and overall survival (OS) following atezolizumab monotherapy in IMvigor210 (HR: 1.34, 95% CI:1.03, 1.74, P=0.026). f, Kaplan-Meier curves depict overall survival of IL8 expression in the tumors of IMmotion using median cutoff in atezolizumab, atezolizumab + bevacizumab and sunitinib arms. High vs. low intratumoral IL8 gene expression and OS with atezolizumab monotherapy (HR: 3.97, 95% CI:1.83, 8.6, P=1.40e-4), atezolizumab + bevacizumab (HR: 1.78, 95% CI: 0.92, 3.4, P= 0.088), and sunitinib (HR: 1.75, 95% CI: 0.83, 3.7, P= 0.144) in IMmotion 150. g, High versus low intratumoral IL8 gene expression and OS in Teff-high patients who received atezolizumab monotherapy in IMvigor210 (HR: 1.67, 95% CI: 1.12, 2.49, P=0.012). h, Kaplan-Meier curves depict overall survival of IL8 expression in the tumors of IMmotion using median cutoff in atezolizumab, atezolizumab + bevacizumab and sunitinib arms. High versus low intratumoral IL8 gene expression and OS in the T-effector (Teff) high patient subset treated with: atezolizumab monotherapy (HR: 15.6, 95% CI: 3.15, 77.6, P=4.70e-4), atezolizumab + bevacizumab (HR: 0.96, 95% CI: 0.29, 3.2, P=0.945), and sunitinib (HR: 1.94, 95% CI: 0.67, 5.6, P=0.225) in IMmotion150. HRs in Figure 4e-h were calculated using stratified Cox proportional hazard regression models, and P values were calculated using stratified log-rank test. P values were adjusted for multiple comparisons. Multivariate analyses adjusted HRs for age, sex, race, ECOG performance status, presence of liver metastasis, and tumor burden (sum of longest diameter, SLD) in mUC; and age, sex, Memorial Sloan Kettering Cancer Risk (MSKCC) prognostic risk score, previous nephrectomy, and SLD in mRCC data sets.