Contribution of systemic and somatic factors to clinical response and resistance to PD-L1 blockade in urothelial cancer: An exploratory multi-omic analysis

<div><p>Background</p><p>Inhibition of programmed death-ligand 1 (PD-L1) with atezolizumab can induce durable clinical benefit (DCB) in patients with metastatic urothelial cancers, including complete remissions in patients with chemotherapy refractory disease. Although mutation load and PD-L1 immune cell (IC) staining have been associated with response, they lack sufficient sensitivity and specificity for clinical use. Thus, there is a need to evaluate the peripheral blood immune environment and to conduct detailed analyses of mutation load, predicted neoantigens, and immune cellular infiltration in tumors to enhance our understanding of the biologic underpinnings of response and resistance.</p><p>Methods and findings</p><p>The goals of this study were to (1) evaluate the association of mutation load and predicted neoantigen load with therapeutic benefit and (2) determine whether intratumoral and peripheral blood T cell receptor (TCR) clonality inform clinical outcomes in urothelial carcinoma treated with atezolizumab. We hypothesized that an elevated mutation load in combination with T cell clonal dominance among intratumoral lymphocytes prior to treatment or among peripheral T cells after treatment would be associated with effective tumor control upon treatment with anti-PD-L1 therapy. We performed whole exome sequencing (WES), RNA sequencing (RNA-seq), and T cell receptor sequencing (TCR-seq) of pretreatment tumor samples as well as TCR-seq of matched, serially collected peripheral blood, collected before and after treatment with atezolizumab. These parameters were assessed for correlation with DCB (defined as progression-free survival [PFS] >6 months), PFS, and overall survival (OS), both alone and in the context of clinical and intratumoral parameters known to be predictive of survival in this disease state.</p><p>Patients with DCB displayed a higher proportion of tumor-infiltrating T lymphocytes (TIL) (<i>n</i> = 24, Mann-Whitney <i>p</i> = 0.047). Pretreatment peripheral blood TCR clonality below the median was associated with improved PFS (<i>n</i> = 29, log-rank <i>p</i> = 0.048) and OS (<i>n</i> = 29, log-rank <i>p</i> = 0.011). Patients with DCB also demonstrated more substantial expansion of tumor-associated TCR clones in the peripheral blood 3 weeks after starting treatment (<i>n</i> = 22, Mann-Whitney <i>p</i> = 0.022). The combination of high pretreatment peripheral blood TCR clonality with elevated PD-L1 IC staining in tumor tissue was strongly associated with poor clinical outcomes (<i>n</i> = 10, hazard ratio (HR) (mean) = 89.88, HR (median) = 23.41, 95% CI [2.43, 506.94], <i>p</i>(HR > 1) = 0.0014). Marked variations in mutation loads were seen with different somatic variant calling methodologies, which, in turn, impacted associations with clinical outcomes. Missense mutation load, predicted neoantigen load, and expressed neoantigen load did not demonstrate significant association with DCB (<i>n</i> = 25, Mann-Whitney <i>p</i> = 0.22, <i>n</i> = 25, Mann-Whitney <i>p</i> = 0.55, and <i>n</i> = 25, Mann-Whitney <i>p</i> = 0.29, respectively). Instead, we found evidence of time-varying effects of somatic mutation load on PFS in this cohort (<i>n</i> = 25, <i>p</i> = 0.044). A limitation of our study is its small sample size (<i>n</i> = 29), a subset of the patients treated on IMvigor 210 (NCT02108652). Given the number of exploratory analyses performed, we intend for these results to be hypothesis-generating.</p><p>Conclusions</p><p>These results demonstrate the complex nature of immune response to checkpoint blockade and the compelling need for greater interrogation and data integration of both host and tumor factors. Incorporating these variables in prospective studies will facilitate identification and treatment of resistant patients.</p></div

Associations between measured somatic, immune, and clinical variables.

<p>(A) Although both programmed death-ligand 1 (PD-L1) staining and mutation load each were weakly associated with response, these variables were not correlated with each other (<i>n</i> = 25, Spearman rho = 0.14 <i>p</i> = 0.51). (B) Pretreatment peripheral T cell receptor (TCR) clonality did not correlate with mutation load (<i>n</i> = 25, Pearson r = 0.0017 <i>p</i> = 0.99). (C) Tumor-infiltrating T lymphocytes (TIL) proportion as estimated by TCR sequencing was associated with PD-L1 immune cell (IC) staining (<i>n</i> = 24, Spearman rho = 0.51 <i>p</i> = 0.010). (D) TIL clonality was associated with PD-L1 IC staining (<i>n</i> = 24, Spearman rho = 0.48 <i>p</i> = 0.017). (E) Hazard associated with log(pretreatment peripheral TCR clonality) by level of IC PD-L1 expression (IC0, IC1, or IC2). (F) Multivariate survival analysis of various clinical, peripheral, and intratumoral biomarkers for association with time to disease progression or mortality, utilizing a varying-coefficient model, which allows the hazard associated with a 1-unit increase in a biomarker’s value to vary according to the level of intratumoral PD-L1 expression (IC score).</p

Time-dependent relationship between mutation load and treatment response.

<p>(A) There was a stronger association between somatic mutation load and progression-free survival (PFS) for events occurring more than 3 months following therapy (blue box: hazard ratio [HR] = 0.69, 95% CI [0.38, 0.99]), as compared to those in the first 3 months (red box: HR = 0.91, 95% CI [0.75, 1.07]). (B) There was a stronger association between somatic mutation load and overall survival (OS) for events occurring more than 3 months following therapy (blue box: HR = 0.80, 95% CI [0.60, 1.00]), as compared to those in the first 3 months (red box: HR = 1.02, 95% CI [0.79, 1.22]). (C) Kaplan-Meier estimates for PFS among patients with missense single nucleotide variants (SNVs) per megabase above the median observed value of 1.03 (red) and those with counts below the median value (blue). Time (Days) is plotted on a log-scale. For context, the frequency of observed events (progression and/or mortality) is plotted below the <i>x</i>-axis among patients with missense SNV per megabase above (red) and below (blue) the median value, with the approximate schedule of follow-up scans per the study protocol (see <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1002309#sec008" target="_blank">Methods</a>) shown as vertical dotted lines.</p

Baseline characteristics.

<p>Baseline characteristics.</p

T cell receptor (TCR) clonality and treatment response.

<p>(A) Tumor-infiltrating T lymphocytes (TIL) proportion alone was associated with durable clinical benefit (DCB), with a median of 0.21 (range 0.049–0.33) in tumors from patients who had DCB versus 0.069 (range 0.0098–0.24) in tumors from patients who did not (<i>n</i> = 24, Mann-Whitney <i>p</i> = 0.047). TIL clonality alone was not significantly associated with DCB, with a median of 0.12 (range 0.047–0.34) in tumors from patients with DCB and a median of 0.092 (range 0.033–0.22) in tumors from patients without DCB (<i>n</i> = 24, Mann-Whitney <i>p</i> = 0.10). (B) Tumors with less than the median TIL proportion or TIL clonality, considered jointly as 1 feature, were less likely to display DCB (Fig 1B, 25% of patients with DCB versus 81% of patients without DCB, <i>n</i> = 24, Fisher's Exact <i>p</i> = 0.021). (C) Patients with pretreatment peripheral TCR clonality less than the median exhibited improved progression-free survival (PFS, <i>n</i> = 29, log-rank <i>p</i> = 0.048). (D) Patients with pretreatment peripheral TCR clonality less than the median exhibited improved overall survival (OS, <i>n</i> = 29, log-rank <i>p</i> = 0.011). (E) There was a significant association between TCR clonality in the peripheral blood prior to initiating treatment and overall survival greater than 12 months (DCB-OS) (DCB-OS: TCR clonality 0.060 [range 0.022–0.21]; OS less than 12 months: 0.15 [range 0.031–0.35, <i>n</i> = 29, Mann-Whitney <i>p</i> = 0.0061]). (F) There was no significant association between pretreatment peripheral TCR clonality and DCB (DCB: TCR clonality 0.068 [range 0.027–0.21]; no DCB: 0.14 [range 0.022–0.35, <i>n</i> = 29, Mann-Whitney <i>p</i> = 0.25]). (G) Expansion of TCR clones found in TIL (orange bars) occurred in the peripheral blood 3 weeks after initiating treatment in all patients. (H) The number of TCR clones found in TIL that expanded in the peripheral blood 3 weeks after initiating treatment was 8.00 (range 4.00–12.00) in patients with DCB and 2.50 (range 1.00–18.00) in non-DCB patients (<i>n</i> = 22, Mann-Whitney <i>p</i> = 0.022).</p