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Purpose: Immune checkpoint inhibitors (ICI) have revolutionized the treatment of non–small cell lung cancer (NSCLC), but predictive biomarkers of their efficacy are imperfect. The primary objective is to evaluate circulating immune predictors of pembrolizumab efficacy in patients with advanced NSCLC. Experimental Design: We used high-dimensional mass cytometry (CyTOF) in baseline blood samples of patients with advanced NSCLC treated with pembrolizumab. CyTOF data were analyzed by machine-learning algorithms (Citrus, tSNE) and confirmed by manual gating followed by principal component analysis (between-group analysis). Results: We analyzed 27 patients from the seminal KEYNOTE-001 study (median follow-up of 60.6 months). We demonstrate that blood baseline frequencies of classical monocytes, natural killer (NK) cells, and ICOS+ CD4+ T cells are significantly associated with improved objective response rates, progression-free survival, and overall survival (OS). In addition, we report that a baseline immune peripheral score combining these three populations strongly predicts pembrolizumab efficacy (OS: HR = 0.25; 95% confidence interval = 0.12–0.51; P < 0.0001). Conclusions: As this immune monitoring is easy in routine practice, we anticipate our findings may improve prediction of ICI benefit in patients with advanced NSCLC.

We retrospectively analyzed non–small cell lung cancer (NSCLC) patients from a single center treated with pembrolizumab on the KEYNOTE-001 trial and evaluated the association between treatment-related adverse events (trAEs) and clinical outcomes. Investigators reported AEs on trial and graded them according to Common Terminology Criteria for Adverse Events v4.0, labeling them as unlikely, possibly, or probably treatment-related. AEs labeled as possibly/probably related were considered trAEs for this analysis. The relationship between the incidence of a trAE and clinical outcomes was evaluated. Ninety-seven NSCLC patients treated on KEYNOTE-001 at the University of California, Los Angeles were evaluated. Ten percent (85/826) of AEs were trAEs, occurring in 40% (39/97) of patients. The most frequent trAEs were rash (21% patients), fatigue (6% patients), and hypothyroidism (6% patients). The 39 patients that experienced a trAE had increased objective response rate (ORR, 38.5%), progression-free survival (PFS: median, 248 days), and overall survival (OS: median, 493 days), compared with the 58 patients that did not (ORR: 8.9%, PFS: median 60 days, OS: median 144.5 days). The observed association between trAEs and improved clinical outcome persisted when using Cox proportional hazards regression models to assess the confounding effect of covariates and mitigate guarantee-time bias. The association also remained when data were substratified by grade, degree of association, and treatment-related select AE designation. This single-center analysis revealed that trAEs predicted for improved clinical outcome with pembrolizumab, and when controlling for guarantee-time bias and plausible confounders, this association remained. This observed relationship adds to our understanding of anti–PD-1 therapy and could aid clinicians in identifying patients most likely to benefit from therapy. Cancer Immunol Res; 6(3); 288–94. ©2018 AACR.

Table S2: Demographics of the 97 NSCLC patients treated at UCLA on KEYNOTE-001 (N=97) compared with the demographics of the total KEYNOTE-001 study population (n=495). Of note, the UCLA cohort of 97 patients is included in the total KEYNOTE-001 study population of 495 patients.

Table S3: The occurrence of a treatment related AE (trAE) in the 97 patients treated at UCLA on the KEYNOTE-001 clinical trial, stratified by a history of thoracic radiation therapy (TRT) or prior radiation therapy to any location. P values computed via Fisher's exact test.

Supplementary Table S6a: Objective response rate (ORR) in the 95 NSCLC patients from the UCLA cohort that had at least 1 set of thyroid values obtained on trial available for analysis stratified by the presence or absence of an abnormal TSH value of varying qualities (Any, High, Low, Both High and Low noted).

Supplementary Table S5a: Cox proportional hazards models for progression-free survival (PFS) with number of treatment related select adverse events included as a time-varying covariate. Listed are results from both unadjusted models and adjusted models. Hazard ratio estimates marked '(+1)' correspond to the incremental effect of a unit increase in the relevant predictor.

Table S1: All adverse events occurring in > 5% of patients in the UCLA NSCLC cohort of 97 patients treated on KEYNOTE-001.

Supplementary Table 4a: Objective response rate (ORR), progression-free survival (PFS), and overall survival stratified by the incidence of a grade 2 or higher treatment related AE (trAE) or a trAE of varying degrees of attribution (possible or probable). Included are all 97 NSCLC patients treated at UCLA on KEYNOTE-001 with available follow-up data.

Supplementary Figure S1A-B. ORR, PFS, and OS analysis for UCLA NSCLC cohort of KEYNOTE-001 trial. ORR, PFS, and OS for all 97 patients in the UCLA cohort of the KEYNOTE-001 trial stratified by the occurrence of a (A) treatment related adverse event (trAE) or (B) a treatment related select AE.

Purpose:Immune checkpoint inhibitors (ICI) have revolutionized the treatment of non–small cell lung cancer (NSCLC), but predictive biomarkers of their efficacy are imperfect. The primary objective is to evaluate circulating immune predictors of pembrolizumab efficacy in patients with advanced NSCLC. Experimental Design:We used high-dimensional mass cytometry (CyTOF) in baseline blood samples of patients with advanced NSCLC treated with pembrolizumab. CyTOF data were analyzed by machine-learning algorithms (Citrus, tSNE) and confirmed by manual gating followed by principal component analysis (between-group analysis).Results:We analyzed 27 patients from the seminal KEYNOTE-001 study (median follow-up of 60.6 months). We demonstrate that blood baseline frequencies of classical monocytes, natural killer (NK) cells, and ICOS+ CD4+ T cells are significantly associated with improved objective response rates, progression-free survival, and overall survival (OS). In addition, we report that a baseline immune peripheral score combining these three populations strongly predicts pembrolizumab efficacy (OS: HR = 0.25; 95% confidence interval = 0.12–0.51; P < 0.0001).Conclusions:As this immune monitoring is easy in routine practice, we anticipate our findings may improve prediction of ICI benefit in patients with advanced NSCLC.

Supplementary Figure 1: Graphical Abstract Supplementary Figure 2: Overall Survival in NSCLC patients treated with pembrolizumab in KEYNOTE-001 according to EGFR mutation Supplementary Figure 3: Recursive partitioning identified classical monocytes and NK as the main immune populations whose frequency splits patients into distinct patterns of overall survival Supplementary Figure 4: Classical monocytes, NK cells and ICOS+ CD4+ T cells are associated with improved pembrolizumab efficacy in advanced NSCLC patients. Supplementary Figure 5: Gating strategy of the 3 main immune populations associated with pembrolizumab efficacy. Supplementary Figure 6: Comparison side by side of Maxstat cut-offs with cut-off from Younden index for overall survival in the 3 populations of interest. Supplementary Figure 7: Survival outcomes in NSCLC patients treated with pembrolizumab in KEYNOTE-001 according to blood baseline frequency of classical monocytes. Supplementary Figure 8: Baseline immune peripheral score is also associated with PFS in melanoma patients treated with PD-1 inhibitors.

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Supplementary Table Legend

Supplemental Figure 1. Schematic presentation of the analyzed parameters in the correlative cohort per progression survival (PFS). TMB: total mutational burden. PR: partial response. SD: stable disease. PD: progressive disease. â-³: mutation. Supplemental Figure 2. Progression Free Survival (PFS) and subgroup survival analysis of the correlative cohort (N=38) by Log-rank (Mantel-Cox) test. PFS By PD-L1 categories ( 0-49% n=23 vs 50-100% n=9, p75th percentile n=6, p=0.142) PFS By CD8 categories (0-5% n=18 vs 6-25% n=14, p=0.037) PFS By CD4 categories (0-5% n=21 vs 6-25% n=9 vs >25% n=2, p=0.123) Supplemental Figure 3. Association among the four correlative parameters. Supplemental Table 1. Association of the four correlative parameters and with other clinical characteristics. Supplemental Table 2. Correlation of patient characteristics and ORR per irRC (PR vs SD/PD). Supplemental Table 3. Patient characteristics (as categorical variables) in the correlative cohort per prior lines of therapy. Supplemental Table 4. PFS and OS of correlative characteristics (as categorical variables) by year. Supplemental Table 5. Characteristics as continuous variables of long term benefiters (Overall survival > 3 years with no intervening therapies) vs all other patients in the correlative cohort.

Purpose:Several biomarkers have been individually associated with response to PD-1 blockade, including PD-L1 and tumor mutational burden (TMB) in non–small cell lung cancer (NSCLC), and CD8 cells in melanoma. We sought to examine the relationship between these distinct variables with response to PD-1 blockade and long-term benefit.Experimental Design:We assessed the association between baseline tumor characteristics (TMB, PD-L1, CD4, and CD8) and clinical features and outcome in 38 patients with advanced NSCLC treated with pembrolizumab (median follow-up of 4.5 years, range 3.8–5.5 years).Results:PD-L1 expression and CD8 infiltration correlated with each other and each significantly associated with objective response rate (ORR) and progression-free survival (PFS). TMB was independent of PD-L1 and CD8 expression, and trended towards association with ORR and PFS. There was no association between CD4 infiltration and outcomes. Only PD-L1 expression was correlated with overall survival (OS). Among 5 patients with long-term survival >3 years with no additional systemic therapy, PD-L1 expression was the only discriminating feature. The increased predictive value for PFS and OS of composite biomarker inclusive of PD-L1, CD8, CD4, and TMB was limited.Conclusions:In patients with NSCLC treated with PD-1 blockade with long-term follow up, TMB, PD-L1, and CD8 were each associated with benefit from PD-1 blockade. Pretreatment PD-L1 expression was correlated with T lymphocyte infiltration and OS, whereas models incorporating TMB and infiltrating CD4 and CD8 lymphocytes did not substantially add to the predictive value of PD-L1 alone for OS.