Computational algorithm-driven evaluation of monocytic myeloid-derived suppressor cell frequency for prediction of clinical outcomes

Abstract

Evaluation of myeloid-derived suppressor cells (MDSC), a cell type implicated in T-cell suppression, may inform immune status. However, a uniform methodology is necessary for prospective testing as a biomarker. We report the use of a computational algorithm-driven analysis of whole blood and cryopreserved samples for monocytic MDSC (m-MDSC) quantity that removes variables related to blood processing and user definitions. Applying these methods to samples from patients with melanoma identifies differing frequency distribution of m-MDSC relative to that in healthy donors. Patients with a pretreatment m-MDSC frequency outside a preliminary definition of healthy donor range (<14.9%) were significantly more likely to achieve prolonged overall survival following treatment with ipilimumab, an antibody that promotes T-cell activation and proliferation. m-MDSC frequencies were inversely correlated with peripheral CD8(+) T-cell expansion following ipilimumab. Algorithm-driven analysis may enable not only development of a novel pretreatment biomarker for ipilimumab therapy, but also prospective validation of peripheral blood m-MDSCs as a biomarker in multiple disease settings.

Trial registration: ClinicalTrials.gov NCT00495066 NCT00920907.

Figure 1. Analysis of Myeloid Derived Suppressor Cell Frequency

Peripheral blood mononuclear cells (PBMC) from advanced melanoma patients and healthy donors were stained with surface antibody and analyzed by multicolor flow cytometry. We defined monocytic myeloid cells based on presence of CD14, CD11b in a CD3, CD16, CD19, CD20, CD56 in lineage (Lin) negative population. Within this monocytic cell population, m-MDSC were isolated based on their low levels of expression of HLA-DR expression. (A). Gating strategy to isolate myeloid derived cells as CD14+CD11b+Lineage- cells. Based on the 99%ile of healthy donor values, a cut-off for low expression of HLA-DR was set to isolate the population of m-MDSC (shaded in red). (B) m-MDSC composition by HLA-DR GMFI is subject to fluctuations in staining acquisition and sample handling. CV_HLA-DR represents a self-normalizing measurement and is stable among replicate measurements. (C). Comparison of coefficient of variations (CV) for HLA expression within the myeloid compartment reveals a larger spread for patients pre-treatment, compared to healthy donors and large differences in CV between patients (HD vs. Patients: p<0.05). (D) Normogram plotting relationship between CV values and m-MDSC frequency. (E) Evaluation of whole blood collected in standard heparin or Cyto-Chex^®tubes (n=9) for m-MDSC frequency and stored at room temperature for the specified interval between analysis and acquisition. Data is expressed as a percentage of total m-MDSC present at baseline. *p=0.002 (F) Correlation between m-MDSC analysis of samples (n=8) cryopreserved using BD Vacutainer^® CPT tubes, standard heparin tubes and collected in Cyto-Chex^®tubes.

Figure 2. Functionally Suppressive m-MDSC are increased in patients with metastatic melanoma less likely to achieve prolonged overall survival following ipilimumab.

(A) Peripheral blood mononuclear cell (PBMC) from advanced melanoma patients and healthy donors analyzed for %m-MDSC based on CV_HLA-DR. The frequency of m-MDSC in healthy donors (n=20), melanoma patients analyzed at pre-treatment baseline and week 6 (Healthy donor vs pretreatment p=0.05; healthy donor vs week 6, p=0.03), (B) Pre-treatment values for subsets of patients treated with ipilimumab 10mg/kg (n=28) or 3mg/kg (n=40). (C) Overall survival based on m-MDSC quantity at pre-treatment baseline. (D) Overall survival from 6 weeks after start of ipilimumab treatment. (E) The correlation between %change in CD8 T cells and wk 6 m-MDSC frequency (r = − 0.541, p = 0.02). % change in CD8 T cells = [(wk6 absolute CD8 – baseline absolute CD8) ÷ (baseline absolute CD8)]. (F) The average stimulation index is graphed for 2 melanoma patients with clinical benefit and 2 melanoma patients with non-clinical benefit assessed at week 24. Stimulation index = (% proliferated CD3+ T cells in CD14-depleted PBMCs) / (% proliferated CD3+ T cell in CD14-PBMCs with CD14+ cells added back).