Article Figures & Data
Figures
- Figure 1.
Analysis of MDSC frequency. PBMCs from patients with advanced melanoma and from healthy donors were stained with surface antibody and analyzed by multicolor flow cytometry. We defined monocytic myeloid cells based on the presence of CD14, CD11b in a CD3, CD16, CD19, CD20, and CD56 in a Lin− population. Within this monocytic cell population, m-MDSCs were isolated on the basis of their low levels of HLA-DR expression. A, gating strategy to isolate myeloid-derived cells as CD14+CD11b+Lin− cells. On the basis of the 99th percentile of healthy donor values, a cutoff for low expression of HLA-DR was set to isolate the population of m-MDSC (shaded in gray). B, m-MDSC composition by HLA-DR GMFI is subject to fluctuations in staining acquisition and sample handling. CVHLA-DR represents a self-normalizing measurement and is stable among replicate measurements. C, comparison of CV for HLA expression within the myeloid compartment reveals a larger spread for patients pretreatment, compared with healthy donors and large differences in CV between patients (healthy donors 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 are expressed as a percentage of total m-MDSCs 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-MDSCs are increased in patients with metastatic melanoma who are less likely to achieve prolonged OS following ipilimumab. A, PBMCs from patients with advanced melanoma and from healthy donors analyzed for %m-MDSC based on CVHLA-DR. The frequency of m-MDSC in healthy donors (n = 20) and patients with melanoma analyzed at pretreatment baseline and week 6 (healthy donors vs. pretreatment, P = 0.05; healthy donors vs. week 6, P = 0.03). B, pretreatment values for subsets of patients treated with ipilimumab 10 mg/kg (n = 28) or 3 mg/kg (n = 40). C, OS based on m-MDSC quantity at pretreatment baseline. D, OS from 6 weeks after start of ipilimumab treatment. E, correlation between percentage change in CD8 T cells and week 6 m-MDSC frequency (r = −0.541; P = 0.02). Percentage change in CD8 T cells = [(wk 6 absolute CD8 − baseline absolute CD8)/(baseline absolute CD8)]. F, average SI graphed for 2 patients with melanoma with clinical benefit and 2 patients with melanoma with nonclinical benefit assessed at week 24. SI = (% proliferated CD3+ T cells in CD14-depleted PBMCs)/(% proliferated CD3+ T cells in CD14-PBMCs with CD14+ cells added back).
Tables
- Table 1.
Patient and healthy donor characteristics
Characteristics Ipilimumab 10 mg/kg Ipilimumab 3 mg/kg Healthy donorsa Number of patients 28 40 20 Median age (range) 62 (34–83) 60 (34–80) 38 (26–58) Sex (%) Male 17 (61) 29 (73) 10 (50) Female 11 (39) 11 (27) 7 (35) Stage of disease (%) III (unresectable) 0 1 — M1a 3 0 — M1b 4 5 — M1c 21 34 — Median number of prior therapies (range) 1 (0–3) 1 (0–5) — Median LDH (range) 209 (113–968) 211 (117–816) — ≥Upper limit of normal (% of available LDH) 13 (46) 28 (70) — <Upper limit of normal (% of available LDH) 15 (54) 12 (30) — MDSC frequency %HLA-DRlow/− in Lin−CD14+CD11b+ (range)b 11.4 (3.9–24.5) 11.2 (5.8–20.9) 10.3 (6.4–14.3) ≥14.9 (%) 7 (25) 7 (18) 0 (0) <14.9 (%) 21 (75) 33 (82) 20 (100) Median baseline ALC (range) 1,250 (500–5,100) 1,100 (600–8,100) — ≥1,000/μL (%) 19 (68) 25 (63) — <1,000/μL (%) 9 (32) 15 (37) — - Table 2.
Univariate analysis of relationship between m-MDSC and OS at pretreatment baseline and week 6 after ipilimumab
Ipilimumab treated Pretreatment Week 6 n HR (95% CI) P n HR (95% CI) P MDSC < 14.9% 68 0.35 (0.18–0.70) 0.002 64 0.38 (0.19–0.75) 0.004 ALC ≥ 1,000 cells/μL 68 0.73 (0.41–1.33) 0.303 64 0.22 (0.11–0.45) <0.001 LDH < 250 68 0.33 (0.18–0.59) <0.001 65 0.37 (0.20–0.68) 0.001 Monocytes < 300 cells/μL 68 0.70 (0.25–1.95) 0.495 64 1.77 (0.69–4.51) 0.233 - Table 3.
Multivariate analysis of relationship between m-MDSC and OS at pretreatment baseline and week 6 after ipilimumab treatment
Ipilimumab Pretreatment Week 6 n HR (95% CI) P n HR (95% CI) P MDSC ≤ 14.9% 68 0.47 (0.23–0.94) 0.033 63 0.38 (0.18–0.81) 0.012 ALC ≥ 1,000 cells/μL — — — 63 0.21 (0.10–0.46) <0.001 LDH < 250 68 0.38 (0.21–0.69) 0.002 63 0.29 (0.15–0.56) <0.001
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure Legends - PDF file - 81K
- Supplementary Figure 1 - PDF file - 142K, Supplemental Figure 1. m-MDSC gating strategy using various HLA-DR cutoffs based on expression of HLA-DR in lineage positive cells yields differing m-MDSC frequencies and is susceptible to inter-user variability.
- Supplementary Figure 2 - PDF file - 58K, Supplemental Figure 2. PBMCs available in multiple aliquots were serially analyzed over 3 weeks to evaluate day-to-day reproducibility of CVHLA-DR measures. A standard error of measurement of 1.4% and 0.7%, respectively for QC sample 1 and 2 was detected.
- Supplementary Figure 3 - PDF file - 88K, Supplemental Figure 3. PBMCs from metastatic melanoma patients treated with ipilimumab depleted of CD14-expressing cells were stimulated to proliferate with OKT-3 and IL-2. CFSE dilution of CD3+ T cells in the culture is measured in the absence of CD14+ cells or with CD14+ cells added back.
- Supplementary Figure 4 - PDF file - 101K, Supplemental Figure 4. Different summary statistics can be applied to measure HLA-DR expression on lineage negative, CD14+CD11b+ cells and assess relationship to overall survival (using maximum logrank statistics as described in Methods). CV was used as a self-normalizing measurement that was preferred to eliminate non-biological variation in clinical measurements (e.g. day-to-day variation, differences in sample handling and FACS acquisition).
- Supplementary Table 1 - PDF file - 103K, Supplemental table 1. Univariate analysis of relationship between m-MDSC and overall survival at pre-treatment baseline and week 6 after ipilimumab treatment at 10mg/kg and 3mg/kg.
Volume 2, Issue 8
