Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma

Abstract

Background: Immune checkpoint blockade is revolutionizing therapy for advanced cancer, but many patients do not respond to treatment. The identification of robust biomarkers that predict clinical response to specific checkpoint inhibitors is critical in order to stratify patients and to rationally select combinations in the context of an expanding array of therapeutic options.

Methods: We performed multiparameter flow cytometry on freshly isolated metastatic melanoma samples from 2 cohorts of 20 patients each prior to treatment and correlated the subsequent clinical response with the tumor immune phenotype.

Results: Increasing fractions of programmed cell death 1 high/cytotoxic T lymphocyte-associated protein 4 high (PD-1hiCTLA-4hi) cells within the tumor-infiltrating CD8+ T cell subset strongly correlated with response to therapy (RR) and progression-free survival (PFS). Functional analysis of these cells revealed a partially exhausted T cell phenotype. Assessment of metastatic lesions during anti-PD-1 therapy demonstrated a release of T cell exhaustion, as measured by an accumulation of highly activated CD8+ T cells within tumors, with no effect on Tregs.

Conclusions: Our data suggest that the relative abundance of partially exhausted tumor-infiltrating CD8+ T cells predicts response to anti-PD-1 therapy. This information can be used to appropriately select patients with a high likelihood of achieving a clinical response to PD-1 pathway inhibition.

Funding: This work was funded by a generous gift provided by Inga-Lill and David Amoroso as well as a generous gift provided by Stephen Juelsgaard and Lori Cook.

Figure 1. Relative abundance of CTLA-4^hiPD-1^hi CTLs predicts response to anti–PD-1 therapy.

(A) Flow cytometric data from metastatic tumors taken prior to anti–PD-1 therapy and representative pre- and post-treatment computed tomographic images from a patient who achieved a response (Responder) and one who did not (Nonresponder). Flow cytometric plots were pregated on live CD45⁺CD3⁺CD8⁺ cells. (B) Discovery cohort (n = 20 patients) and (C) validation cohort (n = 20 patients) of PFS for patients who had 20% or more (dotted line) or 20% or fewer (solid line) tumor-infiltrating CTLA-4^hiPD-1^hi CTLs. Statistical significance was determined by log-rank test.

Figure 2. Clinical characteristics and CTL profiles of patients with metastatic melanoma who responded or did not respond to anti–PD-1 therapy.

Histogram showing flow cytometric quantification of the percentages of CTLA4^hiPD-1^hi tumor-infiltrating T cells in the CD8⁺ CTL gate versus characteristics of individual patients. Responders included patients with tumor target lesions that met RECIST 1.1 criteria for a CR (>99% reduction in the target lesions) or a PR (≥30% reduction in target lesions). Nonresponders included patients with tumor target lesions that met RECIST 1.1 criteria for progressive (≥20% increase in the target lesions) or stable disease (<30% reduction or <20% increase in tumor target lesions). n = 40 patients. Tregs were defined as FOXP3^hiCTLA-4^hi cells within the live CD45⁺CD3⁺CD4⁺ gate. V, validation; D, discovery; I, ipilimumab; T, targeted therapy with BRAF/MEK inhibitors; Liv, liver; LN, lymph node; Mets, metastasis; S, skin; Ova, ovary.

Figure 3. Tumor-infiltrating CTLA-4^hiPD-1^hi CTLs have a partially exhausted phenotype.

(A) Representative flow cytometric plots (pregated on live CD45⁺CD3⁺CD8⁺ cells) of intracellular cytokine staining of tumor-infiltrating lymphocytes from metastatic tumors taken prior to anti–PD-1 therapy. (B) Quantification of cytokine-expressing cells (as measured by intracellular cytokine staining) obtained from multiple metastatic tumors taken prior to anti–PD-1 therapy. White squares and red dots represent individual patients. Bars indicate the mean values. Data were combined from 3 replicate experiments. P value in B was determined by an unpaired, 2-tailed Student’s t test.

Figure 4. Anti–PD-1 treatment results in preferential activation of tumor-infiltrating CD8⁺ CTLs.

(A) Flow cytometric quantification of tumor-infiltrating CD4⁺ and CD8⁺ T cells obtained before and after starting anti–PD-1 therapy. (B) Representative flow cytometric plots and flow cytometric quantification of HLA-DR expression on tumor-infiltrating CD8⁺ T cells before and after starting anti–PD-1 therapy. (C) Flow cytometric quantification of tumor-infiltrating Tregs as well as Treg expression of CTLA-4 and HLA-DR before and after starting anti–PD-1 therapy. White squares and red dots represent individual patients. Bars indicate the mean values. MFI, mean fluorescence intensity. P values in all panels were determined by an unpaired, 2-tailed Student’s t test.